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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:
16 June 2017 - 26 June 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
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
Chemical structure
Reference substance name:
Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatesilicate
EC Number:
EC Name:
Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatesilicate
Cas Number:
Molecular formula:
C54H58N4O6.MoO4 + C54H58N4O6.SiO3
Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatesilicate
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: pink powder
Storage conditions: at room temperature
Specific details on test material used for the study:
- Stability in water: stable
- Correction factor for purity: 1.074 (concentrations in the report are expressed as weight test item per volume)

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples were taken from the highest test concentration and the control according to the schedule below.
Frequency: at t=0 h and at t=48 h
Volume: 2.0 mL from the approx. 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 application to the test medium: preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. An overnight period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of filtration through a 0.45 µm membrane filter and used as test concentrations.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material: The highest WAF was clear and slightly pink, while all remaining test solutions were clear and colorless at the end of the preparation procedure. No Tyndall effect was observed in any of the test solutions when they were observed with a laser pen.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia
- Age at study initiation: <24 hours
- Source: in-house laboratory culture with a known history, originating 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.
- Age of parental stock: >2 weeks
- Feeding during test: no

- Breeding method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Cultures (maximum age of 4 weeks) were renewed after 7 days of cultivation by renewing the medium twice a week.
- Feeding: daily, a suspension of fresh water algae.
- Health during breeding period (any mortality observed): no

Study design

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

Test conditions

180 mg CaCO3/L
Test temperature:
20 - 21°C
Start: 7.7-7.9
End: 8.1
Dissolved oxygen:
Start: 8.9 - 9.0
End: 8.7 - 8.8
Nominal and measured concentrations:
Nominal concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
Mean measured concentration: 0.067 mg/L was calculated to be the geometric mean exposure concentration in the WAF prepared at 100 mg/L.
Measured concentration at t=0 h: 0.079 mg/L; measured concentration at t=48 h: 0.057 mg/L (73% of initial)
Details on test conditions:
- Test vessel: 60 mL, all-glass vessels, open, fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest test concentration, 2 for each intermediate test concentration.
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes, M7 medium
- Intervals of water quality measurement: at t=0 h and at t=48 h (dissolved oxygen and pH measurements)

- Adjustment of pH: no
- Photoperiod: 16 hours daily

EFFECT PARAMETERS MEASURED: immobility at t=24 and t=48 hours

- Test concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: not applicable, the limit test was reported as the definitive study
Reference substance (positive control):
potassium dichromate (June 2017)

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
> 0.067 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
Details on results:
- No immobility was recorded in the control or at any of the test concentrations.
- Abnormalities observed: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, the test solutions were observed with a laser pen for a Tyndall effect which had a negative result.
- Effect concentrations exceeding solubility of substance in test medium: yes, the EC50 could not be reached at the maximum dissolution of the highest test concentration.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Concentrations tested: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- Relevant effect levels: 48h-EC50: 0.59 mg/L, 95% CI: 0.52-0.66 mg/L
- Other: the results where within the expected range according to the historical data (>0.3 and <1.0 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:
see 'overall remarks'
The 48h-EC50 for Daphnia magna exposed to C.I. Pigment Red 81:2 exceeded an average measured concentration of 67 µg/L, which was considered to be the maximum soluble concentration of the test item in test medium.
Executive summary:

In a short-term toxicity test, performed according to OECD guideline 202 and GLP principles, C.I. Pigment Red 81:2 was assessed for its potential toxicity towards Daphnia magna. The test item was poorly soluble and therefore test solutions were individually prepared at loading rates of 1.0, 10 and 100 mg/L, followed by magnetic stirring overnight to ensure maximum dillution of the test item in medium. Water Accomodated Fractions (WAFs) were collected by filtering the test solutions and used as test concentrations. Samples of the blank control and the highest test concentration were taken for analysis at the start and at the end of the test.

Twenty daphnids were exposed to the untreated control and the highest test concentration whereas ten daphnids were exposed to the intermediate test concentrations. The exposure period was 48 hours under static conditions.

Analysis of the test solutions showed that the highest test concentration was 0.076 mg/L at t=0 and 0.057 mg/L at t=48 (73% of initial). Based on these results, the geometric mean exposure concentration was calculated to be 0.067 mg/L. No immobility was observed at any of the tested concentrations. Therefore, the 48h-EC50 for Daphnia magna exposed to C.I. Pigment Red 81:2 was determined to be >67 µg/L, which was considered to be the maximum soluble concentration of the test item in test medium.

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

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