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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2017 - 18 October 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
according to guideline
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
GLP compliance:

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white powder with lumps
- Test item storage: at room temperature protected from light
Specific details on test material used for the study:
pH (1% in water, indicative range): 5.12 – 4.78
Solubility in water: poor
Stability in water: not indicated

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control.
In addition, the filter containing the undissolved residue was kept for possible analysis.

Frequency: at t=0 h and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct addition - test solutions with nominal loading rates at 1.0, 10 and 100 mg/L were individually prepared. A 3-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) collected after filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as test concentrations.
A correction was made for the purity/composition of the test item (correction factor of 1.064 was used). All further concentrations reported are based on the pure test item. Weighing and preparation of test solutions was performed under dimmed light.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Strain/clone: at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions
- Source: In-house laboratory culture with a known history
- Age of parental stock: more than two weeks old
- Age of test organisms at test start: < 24 hours
- Feeding during test: no
- Other: 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 reported

- Breeding: Each batch stars with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel at 18-22°C. Daphnids are fed daily, a suspension of fresh water algae. Maximum age of cultures is 4 weeks.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:

Test conditions

180 mg/L (expressed as CaCO3)
Test temperature:
start: 7.7-7.8
end: 8.0
Dissolved oxygen:
start: 8.2 - 8.9 mg O2/L
end: 8.7 mg O2/L
Nominal and measured concentrations:
Combined limit/range-finding test nominal concentrations: control and WAFs individually prepared at nominal loading rates of 1.0, 10 and 100 mg/L
Measured/estimated concentration: in WAF prepared at nominal loading rate of 100 mg/L - 0.40 μg/L (geometric mean measured concentration)
Details on test conditions:
- Test vessel: 60 mL, all-glass
- Fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 1 daphnid/10 mL

- Source/preparation of dilution water: adjusted ISO medium
- Culture medium different from test medium: yes - M7
- Intervals of water quality measurement: temperature - continuously in a temperature control vessel, from start to end of exposure; pH and dissolved oxygen - at the beginning and at the end of the test, for the highest test concentration and the control.

- Adjustment of pH: no
- Photoperiod: no, the test was performed in the dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mobility, including mortality (at t=24 and t=48 h)

- Test concentrations: combined RF/limit test
- Results used to determine the conditions for the definitive study: no definitive study performed
Reference substance (positive control):
potassium dichromate (most recent test performed in september 2017)

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
> 0.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
Remarks on result:
other: EC50 value is considered to be the maximum solubility in medium
Details on results:
- Behavioural abnormalities: none reported
- Other biological observations: none reported
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Dose-response test: yes (0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L)
- EC50: 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L; 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L
- Other: actual responses were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L, and the sensitivity of this batch of D. magna was in agreement with the
historical data collected at the test facility
Reported statistics and error estimates:
No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration).

Any other information on results incl. tables

Analytical verification of test concentrations:

- Measured concentrations in samples taken from the WAF prepared at a nominal loading rate of 100 mg/L were below the limit of quantification.

Therefore actual exposure concentrations were calculated by extrapolation of the calibration curve. The estimated values were 0.63 and 0.26 μg/L at the start and at the end of the test, respectively (i.e. 41% relative to initial).

Based on these results, the average exposure concentration (geometric mean) based on estimated concentrations was 0.40 μg/L.

- Other: Small responses at the retention time of the test item were detected in the chromatograms of the blank QC samples. These responses however were acceptable (maximum contribution to LOQ based on peak area was 6.6%).

Biological effects:

No immobility was observed in the control or at any of the concentrations tested throughout the test duration in this combined limit/range finding test.

Applicant's summary and conclusion

Validity criteria fulfilled:
1) In the control, no daphnids became immobilised or showed other signs of disease or stress. 2) The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
The 48h-EC50 value to Daphnia magna was >0.4 μg/L based on geometric mean measured concentrations.
This concentration is considered to reflect the maximum solubility of the test item in test medium.
Executive summary:

A study was performed to assess the acute toxicity of ROC-118 to Daphnia magna after 48 hours of exposure. The study was conducted in accordance with OECD 202 and GLP, under static conditions. The test item was corrected for purity and test item concentrations were reported as mg pure test item/L.


A combined limit/range-finding study was performed with test solutions prepared individually at nominal loading rates of 1.0, 10 and 100 mg/L (corrected for purity) by applying a 3 -day period of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters, whereafter the Water Accommodated Fractions (WAFs) were used for testing.


Twenty daphnids (4 replicates of 5 daphnids per group) were exposed to a blank control and a WAF prepared at a nominal loading rate of 100 mg/L, and ten daphnids (2 replicates of 5 daphnids per group) were exposed to WAFs prepared at nominal loading rates of 1.0 and 10 mg/L, respectively. Samples taken the untreated control and the limit concentration were analysed for confirmation of actual exposure concentrations at the start of the test and after 48 hours of exposure. The limit test concentration was shown to be below the limit of quantification at test start already, so the exposure concentrations at t=0 and t=48h were estimated by extrapolation from the calibration curve, to be 0.63 and 0.26 μg/L, respectively. From these concentrations, a geometric mean measured concentration of 0.40 μg/L was calculated for the highest concentration tested.

No immobility was observed in the control or at any of the concentrations tested throughout the test duration.

The 48 -hEC50 towards daphnids was >0.40 μg/L, based on the geometric mean measured concentration. This concentration is considered to reflect the maximum solubility of the test item in test medium.

All acceptability criteria were met and the study was considered to be valid.

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