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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:
2016-10-11 to 2016-11-29
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Sampling method: Duplicate samples from each test concentration with surviving daphnids and the control at start of experiment, after 24h, when test medium is renewaled and after 48h (i.e. the end of the study).
- Pre-treatment: Before storage, the samples were extracted with internal standard solution.
- Sample storage conditions before analysis: All samples were deep-frozen at -20°C immediately after sampling.
Details on test solutions:
- Method:
On day 0 and at test medium renewal (day 1): 232 mg in 2320 mL test water mixed intensively for 3 h at room temperature in the dark
After stirring emulsion was sieved over 0.45 µm membrane filter (Whatman NC45).
Precautions during filtering:
The filter was pre-conditioned with filtrate to avoid loss of dissolved item due to adsorption.
A negative pressure was applied on the filtration unit to avoid losses of the volatile test item.
Further dilution of the undiluted filtrate: 1:3.2, 1:10, 1:32, 1:100
Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain/clone: Straus clone
- Source: Daphnia collection University of Basel, Switzerland
- Age of organisms: 6-24h old, not first brood progency
- Food type: algal suspension Desmodesmus subspicatus or a mixture of this algal suspension and a commercial fish diet
- Frequency: 3 times per week
- Test water: reconstituted water

- Acclimation period:No acclimation

Test type:
Water media type:
Renewal of test media after 24h exposure
Limit test:
Total exposure duration:
48 h
2.5 mmol/L
Test temperature:
Dissolved oxygen:
8 mg/L
0.8 mmol/L
Nominal and measured concentrations:
Dilutions of the filtrate: 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate
measured concentrations as arithmetic mean: not analysed (below NOEC), 0.51 mg/L, 1.8 mg/L, 5.9 mg/L and 'not analysed' (all daphnids were dead after 24h)
Details on test conditions:
- Test vessel: glass vessel
- Type: closed, tightly sealed with glass stoppers
- Material, size, headspace, fill volume: No headspace, 60 mL test medium.
- Aeration: None
- Renewal rate of test solution: renewal after 24h
- 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 according to ISO
- Culture medium different from test medium: No

- Photoperiod: 16h light, 8h dark.
- Light intensity: between 15 and 18 μmol m-2 s-1


- Test concentrations: undiluted filtrate (100 mg/L loading rate, filtered over 0.45µm), dilution series 1:10, 1:100 and control.
- Results used to determine the conditions for the definitive study: 100 % immobilization was observed in the undiluted filtrate, but no immobilization at lower concentrations.
Reference substance (positive control):
use of potassium dichromate as positive control, assessment twice per year (latest run: Oct 2016)
Key result
48 h
Dose descriptor:
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
and EC0
Effect conc.:
0.51 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
5.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Behavioural abnormalities: None observed
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 24-h EC50 = 1.0 mg/L
Reported statistics and error estimates:
The 48-hour EC50 value was calculated by Probit Analysis using linear maximum likelihood regression. Calculation of the 95% confidence interval was not possible. Statistical analysis was performed using ToxRat Professional®. The 48-hour NOEC, EC0 and EC100 were determined directly from the raw data.

The concentration of the test samples was assessed at regular intervals:

Treatment 0h-fresh medium (mg/L) 24h (mg/L) 24h, renewal test medium  (mg/L) 48h (mg/L)
control <LOQ  <LOQ  <LOQ  <LOQ
dilution 1:32 0.451 0.5 0.526 0.545
dilution 1:10 1.79 1.97 1.82 1.58
dilution 1:3.2 5.68 5.31 6.8 5.88
undiluted filtrate (100 mg/L loading rate) 19.8 19.6    

<loq 0 0.0284 mg/L

The immobilization of the daphnids was assessed after 24h and 48h.

Treatment mean measured concentration (mg/L) No. Of daphnids tested Immobilized daphnids  % immobilization
      24h 48h 24h 48h
control -- 20 0 0 0 0
dilution 1:100 n.a. 20 0 0 0 0
dilution 1:32 0.51 20 0 0 0 0
dilution 1:10 1.8 20 0 2 0 10
dilution 1:3.2 5.9 20 10 20 50 100
undiluted filtrate (100 mg/L loading rate) 20 20 20 -- 100 --

n.a. Not analyzed since below NOEC

The test performed with spiked samples show a recovery ranging between 92 and 102 %, which hence meets the criteria that recovery should be in the range of 80 -120%.

Validity criteria fulfilled:
The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test according to OECD 202. The 48-hour EC50 was calculated to be 2.3 mg/L (the 95 % confidence limits could not be determined).
Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour test according to the OECD 202.

As the test item is possibly volatile, the test was performed in a closed system using glass tubes without headspace. A semi-static test design with a test medium renewal after 24 hours was used.

Due to the low water solubility of the test item, the test media were prepared following the stirring/filtration method: the test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours in a closed vessel. After stirring, the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the filtrate were used as test media.

For the determination of the actual test item concentrations each test concentration at the start and at the end of the two 24 -hour test medium renewal periods was measured.

At the end of the two renewal periods, the test item concentrations were in the range of 87 to 111 % of the initially measured values. The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of all concentrations measured at the start and the end of the two test medium renewal periods.

Description of key information

For this endpoint, one study assessing the impact of the test item on Daphnia magna is available. The study was conducted according to OECD 202 in a 48h semi-static test, with a test medium renewal after 24h. The daphnids were exposed to undiluted filtrate (100 mg/L loading rate), and a series of dilution of the filtrate: 1:3.2, 1:10, 1:32 and 1:100. The 48h-EC50 value was determined to be 2.3 mg/L (measured, arithmetic mean).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.3 mg/L

Additional information

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