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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31st August 2018 to 20th September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 202 / EC 440/2008.C.2.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Acute Immobilisation Test, adopted April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council, C.2. Daphnia sp. Acute Immobilisation Test (O.J. L 142 of 31.5.2008)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Acid Brown 396:1
- Lot/batch No.: lot No. 77171, ACH sample no. M1807-07566
- Purity: 100%
- Physical form: solid.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition: at room temperature, protected from light
- Solubility: the test item is soluble in test water.
Analytical monitoring:
yes
Remarks:
pH values, O2 conc., conductivity and temperature
Details on sampling:
CONCENTRATIONS
Nominal concentrations were chosen for the definitive test: 100, 50, 25, 12.5 and 6.25 mg/l.

SAMPLING METHOD
The following test vessels were set up:
- Test solution (Tn); containing Daphnia medium with test item (four replicates per concentration with five Daphnia each);
- Control (Bn); containing pure Daphnia medium (four replicates with five Daphnia each).
Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in test water). The resulting solutions were used as the test solutions in the toxicity test. Samples for the determination of the test concentrations were taken from the remaining solutions. Pure test water served as control. O2 concentration and pH were measured in the test medium for each concentration including the control. If the O2 concentration was too low (>5.0 mg/l required) the media were aerated by stirring. Afterwards, the media were filled into the test vessels (50 ml per vessel). Then Daphnia, aged less than 24 h and already acclimatized to the Daphnia medium, were introduced into the test media and the vessels were covered with a glass plate to avoid evaporation and contamination of the test solutions with dust. The Daphnia were not fed during the test and the test vessels were not aerated. After 24 h, new test media (test solutions and control) were prepared as described above and the Daphnia were transferred to these fresh test media.
Vehicle:
yes
Remarks:
Elendt M4 Medium
Details on test solutions:
Reconstituted water (Elendt M4 medium) prepared according to the test guidelines was used for daphnia cultivation and testing. Analytical grade salts were dissolved in sterile purified water. After preparation, the test water was aerated until a sufficient oxygen concentration of ≥3 mg O2/l has been reached.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus),
- Method of breeding: parental and young Daphnia are held in 75 ml glass vessels with 50ml of medium at 18–22 °C, controlled at ±1 °C. One parental Daphnia per vessel.
- Source: derived from a healthy stock and not first brood progeny
- Age: less than 24 hours
- Feeding before test: suspension of the green algae Desmodesmus subspicatus in Elendt M4 medium with an optical density OD680 of about 12 units
- Feeding during test: the Daphnia were not fed during the test
- Illumination: 16h per day
- Control of sensitivity: acute reference test with potassium dichromate conducted twice a year (The EC50 value for the control of sensitivity for 24 h of exposure with potassium dichromate was estimated to be 1.5 mg/l (data from March 2018), which lies within the recommended range of 0.6 – 2.1 mg/l according to OECD Guideline 202.)
Test type:
semi-static
Water media type:
freshwater
Remarks:
Elendt M4 medium
Total exposure duration:
48 h
Hardness:
2.5 mM (based on the sum of Ca2+ and Mg2+), Carbonate Alkalinity: 0.8 mM
Test temperature:
18 – 22 °C, controlled at ± 1 °C
pH:
6 to 9. The pH should normally not vary by more than 1.5 units in one test.
Dissolved oxygen:
7.7< [O2] < 8.0 mg/L
A sufficient oxygen concentration is considered to be >3 mg/L.
Conductivity:
644 [µS*cm^-1]
Nominal and measured concentrations:
MEASURED CONCENTRATIONS
- At the beginning of the test: 94.4, 48.5, 24.3, 13.4 and 6.04 mg/l;
- In the old media after 24 hours of exposure: 102, 48.3, 23.4, 13.6 and 5.37 mg/l (i.e. respectively 102, 97, 94, 109 and 86% of the nominal value).
- After renewal of the test media after 24h (fresh media): 98.6, 49.4, 23.8, 11.8 and 6.01 mg/l;
- After renewal of the test media after 24h (old media): 97.8, 48.9, 24.4, 12.1 and 6.01 mg/l (i.e. respectively 98, 98, 97, 97 and 96% of the nominal value).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml beakers, all-glass, with 50 ml of test medium, covered with a glass plate to avoid evaporation and contamination of the test solutions with dust.
- No. of organisms per vessel: 20 individuals per test concentration and for the blank divided in 4 groups of five individuals

RANGE-FINDING STUDY
Prior to the definitive test, a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of Acid Brown 396:1 (test item as a whole) was performed under static conditions (two replicates for each of the test concentration and the blank control; five daphnids per replicate) to determine a range of concentrations by photometry. The photometry determinations indicated that the test item concentrations significantly decreased at the lower concentrations; which is consistent with the precipitation observed after 48h hours of exposure. Therefore, semi-static conditions were applied in the definitive test with renewal of the test medium after 24h of exposure.
Reference substance (positive control):
not specified
Remarks:
There is a blank of pure Daphnia medium (four replicates with five Daphnia each) and a control of pure test water.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
DETERMINATION OF ECx AND NOEC VALUES AFTER 24H AND 48H
No significant effects (≤10% immobilization) were observed at 100, 50, 25, 12.5 and 6.25 mg/l nominal concentration or in the blank controls after 24 h of exposure.
After 48 h, no significant effects (≤10% immobilization) were observed at 100, 50.0, 25.0 and 12.5 mg/l nominal concentration or in the control. However, 25% immobilization was observed at the nominal concentration of 6.25 mg/l, which cannot be explained.
Therefore, the no-observed effect concentration was not assessed. The median effect concentrations (EC50) of Acid Brown 396:1 on Daphnia magna after 24 h as well as after 48 h of exposure were estimated to be >100 mg/l nominal concentration.

VALIDITY OF THE TEST
The validity criteria were fulfilled:
- In the control, not more than 10% of the daphnids should have been immobilised or show other signs of disease or stress;
- The dissolved oxygen concentration at the end of test should be ≥3 mg O2/l in control and test vessels.
Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 of the test item Acid Brown 396:1 to Daphnia magna was estimated to be greater than 100 mg/l nominal concentration. All validity criteria were fulfilled.
Executive summary:

The acute toxicity of Acid Brown 396:1 to Daphnia magna was investigated according to test guideline OECD 202 under semi-static exposure conditions over a period of 48 h. The test item Acid Brown 396:1 is solid, soluble and is 100% pure. The test media were prepared by respective dilutions of a stock solution with renewal of the test media after 24 h of exposure.The nominal test item concentrations tested were 100, 50.0, 25.0, 12.5 and 6.25 mg/l. Four replicates for each test concentration as well as for the control, were tested. The concentrations of Acid Brown 396:1 in the test media were determined by photometry at the beginning, after 24h (in the fresh and old media) and 48h of exposure.The measured concentrations in the fresh test media (beginning and after 24 hof exposure) were all between 94 and 107% of the nominal values. Thus, the correct dosage of the test item was verified. During each 24h-renewal cycle, the test item concentrations in the test media remained stable and were within 80–120% of the nominal concentrations. Therefore, the biological results were based on the nominal concentrations. After 24h of exposure, no significant number (≤10% immobilization) of immobilized test organisms was observed at any of the test concentrations or in the control. Even after 48 h, no significant effects (≤10% immobilization) were observed at 100, 50.0, 25.0 and 12.5 mg/l nominal concentration or in the control. However, 25% immobilization was observed at the nominal concentration of 6.25 mg/l, which cannot be explained. Therefore, the no-observed effect concentration was not assessed. The biological test results of Acid Brown 396:1to Daphnia magna after 24 and 48 h of exposure are summarized in table, based on the nominal concentrations:

Parameter

Immobilization (0-24 h)

[mg/l]

Immobilization (0-48 h)

[mg/l]

EC50

>100

>100

NOEC

not assessable

not assessable

The 48-hour EC50 of the test item Acid Brown 396:1 to Daphnia magna was estimated to be greater than 100 mg/l nominal concentration. All validity criteria were fulfilled.

Description of key information

The objective of this study was to determine the effect of Acid Brown 396:1 on the mobility of Daphnia Magna according to the OECD guideline 202 under semi-static exposure conditions over a period of 48h. The concentrations of Acid Brown 396:1 in the test media were determined by photometry at the beginning, after 24h (in the fresh and old media) and 48h of exposure. After 24h and 48h, no significant number (≤10% immobilization) of immobilized test organisms was observed at any of the test concentrations or in the control. However, 25% immobilization was observed at the lowest nominal concentration (6.25 mg/l) which cannot be explained. Therefore, the no-observed effect concentration was not assessed. The 48-hour EC50 of the test item Acid Brown 396:1 (EC no.947-779-0) to Daphnia was estimated to be greater than 100 mg/l nominal concentration.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
150 mg/L

Additional information