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Ecotoxicological information

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:
15 Jun - 21 Jul 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
Details on sampling:
- Concentrations: control and 100 mg/L (pooled replicates)
- Sampling method: Duplicate samples were taken at 0 and 48 h.
- Sample storage conditions before analysis: Stored frozen for further analysis.
Details on test solutions:
- Method: 100 mg of the test item was separately added to 1 L of test water to give a nominal loading rate of 100 mg/L. The test water was stirred by a magnetic stirrer using a vortex speed to form a dimple at the water surface. Stirring was stopped after 23 h and the mixture was allowed to stand for 1 h. Microscopic observations indicate a large proportion of undissolved test material. Thus, the solution was filtered through a glass wool plug (2-4 cm length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF was removed by mid-depth siphoning (the first 75-100 mL were discarded). After filtration no micro-dispersions of the test item were present as indicated by microscopic observation.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Source: in-house culture
- Age at study initiation: < 24 h
- Method of breeding: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approx. 20 °C. The lightning cycle was 16 h light and 8 h dark with a 20 min transition period. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 h old.
- Feeding during test: none
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Test temperature:
21 - 22 °C
7.7 - 8.0
Dissolved oxygen:
8.8 - 9.0 mg O2/L
Nominal and measured concentrations:
nominal: control and 100 mg/L (loading rate)
measured: < LOQ (control), 0.0642 mg/L (100 mg/L after 0 h), 0.0255 mg/L (100 mg/L after 48 h)
Details on test conditions:
- Test vessel: glass jars
- Type (delete if not applicable): covered to reduce evaporation
- Material, size, headspace, fill volume: glass, 250 mL, headspace: 50 mL, fill volume: 200 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: reconstituted water (ISO medium) according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded daily. Dissolved oxygen and pH were recorded at the start and termination of the test.

- Photoperiod: 16 h light / 8 h dark
- Light intensity: 573 - 586 lux

- Immobilization and adverse reactions were recorded at 24 and 48 h after start of exposure.

- Range finding study
- Test concentrations: Nominal loading rates of 1, 10 and 100 mg/L (same preparation of test solutions as in the definitive test)
- Results used to determine the conditions for the definitive study: No immobilisation was observed at 1, 10 and 100 mg/L loading WAFs.
Reference substance (positive control):
potassium dichromate
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
other: WAF
Basis for effect:
Details on results:
- Behavioural abnormalities: No sub-lethal effects were observed during exposure.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test solutions appeared to be clear and colorless without undissolved particles after microscopic oberservation.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50/LC50: EC50 (48 h): 0.75 mg/L (95% CI: 0.69 - 0.82 mg/L)
- Other: NOEC (48 h): 0.56 mg/L

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Table 1: Validity criteria for OECD 202 (2004)

Criterion from the guideline


Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

None of the control daphnids showed immobilization or other signs of disease or stress.


The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

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



Validity criteria fulfilled:
See Table 1 in "Any other information on results incl. tables".

Description of key information

No effects up to the limit of water solubility (OECD 202)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the short-term toxicity of Fatty acids, C16-18, esters with diethylene glycol (CAS 85116-97-8) to aquatic invertebrates. Daphnia magna (< 24 h old) was exposed to a nominal loading of 100 mg/L which was prepared as Water Accommodated Fraction to remove any undissolved test material. The actual exposure concentrations were verified by HPLC/MS analysis. 0.0642 mg/L and 0.0255 mg/L were analysed after 0 h and 48 h, respectively.

After exposure of 48 h no immobile daphnids were observed resulting in an EL50 (48 h) > 100 mg/L (nominal). The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
In conclusion the test substance will not exhibit toxic effects to aquatic invertebrates up to the limit of water solubility.