Reaction product of diazotized 3-aminobenzenesulfonic acid coupled with naphthalen-1-amine, subsequently diazotized and coupled with 3-((ethyl(phenyl)amino)methyl)benzenesulfonic acid or 4-((ethyl(phenyl)amino)methyl)benzenesulfonic acid, sodium salt

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

other: read across on similar substance

Adequacy of study:

key study

Study period:

From the 15th of August to the 08th of September, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted according to an internationally accepted test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The temperature was kept at 23-24 °C (instead 18–22 °C), controlled at ±1 °C. This deviation had no influence

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Principles of method if other than guideline:

WSFs (water soluble fractions)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

EC Number:

274-403-7

EC Name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Cas Number:

70210-06-9

Molecular formula:

C31H25N5Na2O6S2

IUPAC Name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the beginning of the test, as well as after 24 and 48 h of exposure.

Test solutions

Vehicle:

yes

Details on test solutions:

Elendt M4 medium
Because the test item is not pure and not well soluble in Daphnia medium, the test solutions were prepared as WSFs (water soluble fractions).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Daphnia magna (Straus, 1820) derived from a healthy stock and not first brood progeny
Age: less than 24 h
TEST SYSTEM
Breeding: parental and young Daphnia held in 4 L glass aquaria (3 L medium) at 20 ± 2 °C
Illumination: 16 h photoperiod a day, supplied by overhead white fluorescent tubes
Medium: continuously aerated Elendt M4 medium; prepared with ultra-pure water (conductivity <1.5 µS/cm)
Feed: Suspension of Desmodesmus subspicatus (green algae) in Elendt M4 medium with an optical density OD680 of about 15 units
Control of sensitivity: acute reference test with potassium dichromate conducted twice a year

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

23 - 24 °C
Determined in an additional flask without Daphnia at the beginning of the test and after 24 and 48 h of exposure.

pH:

6.9 - 7.7
Determined in the combined test solutions for each concentration at the beginning and after 48 h of exposure.

Dissolved oxygen:

5.5 - 7.2 mg/L
O2 concentration and pH were measured in the test medium for each concentration including the blank control. If the O2 concentration was too low (>5.0 mg/l required) the media was aerated by stirring.
Determined in the combined test solutions for each concentration at the beginning and after 48 h of exposure.

Conductivity:

796 - 888 µS.cm-1
Determined in the combined test solutions for each concentration at the beginning of the exposure.

Nominal and measured concentrations:

300, 136.4, 62.0, 28.17, and 12.81mg/L loading rate of test item,
219, 99.8, 45.3, 20.6 and 9.37 mg/l active ingredient.
Higher loading rates than in the range finding test were chosen because 50% effects were not attained in the preliminary test.

Details on test conditions:

Test vessel: 100 ml flasks, all-glass, with 50 ml of test medium, covered with a glass plate to avoid evaporation and contamination of the test solutions with dust
N. Daphia: 40 individuals per test concentration, 20 per vessel
Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes
Feed: the Daphnia are not fed during the test

All values apart from the temperature were within the acceptable range.
The temperature was kept at 23-24°C and therefore not within the required range of 18–22 °C, controlled at ±1°C. This deviation had no influence on the outcome of the test results since no mortality was observed in the blank control and all other validity criteria were fulfilled.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

18.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

PRELIMINARY, NON-GLP TEST
% immobile Daphnia after 48 h of exposure: 17.5 AT 100 mg/L loading rate
DEFINITIVE TEST
The test concentrations during the 48 h test period were measured by HPLC analysis. These analyses confirmed that the test item was only partially soluble in Daphnia medium (20, 17, 13, 12 and 19% of the 9.37, 20.61, 45.3, 99.8 and 219 mg/L loading rate, respectively) and that the concentrations of the test item were satisfactorily maintained over the whole 48 h test period (99, 97, 101, 99 and 97%, respectively, of the initial value).
The geometric mean (GM) of the measured concentrations of the active ingredient (AI) after 24 and 48 h were 1.85, 3.40, 5.94, 12.1 and 42.5 mg/L and 1.85, 3.37, 5.95, 12.0 and 42.1 mg/L, respectively. The effective concentrations ErCx/EyCx were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.
After 24 h of exposure to the test item, following immobilization rates were observed: 62.5% at 219 mg/L, 50% at 99.8 mg/l and 30% at 45.3 mg/L.
No significant effects (≤10% immobilization) were observed at the loading rates of 20.6 and 9.37 mg/l or in the blank controls.
After 48 h of exposure to the test item, following immobilization rates were observed: 87.5% at 219 mg/L, 77.5% at 99.8 mg/L and 55% at 45.3 mg/L.
No significant effects (≤10% immobilization) were observed at the loading rates of 20.6 and 9.37 mg/L or in the blank controls.
Eventhough, the experimental data did not allow for a good statistical fit, they clearly show that the 48 h EC50 to Daphnia magna is <10 mg/l (since 55% immobilization is observed at 5.95 mg/L measured concentration).

Results with reference substance (positive control):

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 0.89 mg/L (data from 29 June 2016), which lies within the recommended range of 0.6–2.1 mg/L according to OECD Guideline 202.

Reported statistics and error estimates:

Statistical analysis was performed with respect to the effect concentrations (including 95% confidence limits)

Any other information on results incl. tables

The effect concentration ECx is the concentration leading to immobilization of x % of the Daphnia after 24 or 48 h of exposure. Those individuals not able to swim within 15 s after gentle agitation of the test vessel were considered to be immobile (even if they were still able move their antennae). The effect concentration ErC50 and EyC50 were determined with ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany).

The no observed effect concentration (NOEC) is the highest concentration tested for which the observed effect is not significantly different from the blank controls. Immobilization rates of <=10% are regarded as not biologically significant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

A test is considered valid if not more than 10% of the Daphnia in the control were immobilized, trapped at the surface of the water, or showed any signs of disease or stress. The dissolved oxygen concentration at the end of the test has to be ≥3 mg O2/L.

Conclusions:

EC50 Daphnia magna (24h): 18.6 mg/L
EC50 Daphnia magna (48h): 8.18 mg/L
based on the geometric mean (GM) of the measured concentrations of the active ingredient.

Executive summary:

Method

The median effect concentration (EC50) and the no-effect concentration (NOEC) of the substance to Daphnia magna were investigated under static exposure conditions over a period of 48 h, following the guideline OECD 202. Since the test item proved to be only partially soluble in Daphnia medium, the test solutions were prepared as WSFs (water soluble fractions). The investigated concentrations were 300, 136, 62.0, 28.2 and 12.8 mg/L loading rate of the test item, corresponding to 219, 99.8, 45.3, 20.6 and 9.37 mg/L of the active ingredient.

40 individual Daphnia divided into 2 test vessels were expose to each concentration of the test item, and 60 Daphnia divided into 3 test vessels were used as blank controls. The test concentrations during the 48 h test period were measured by HPLC analysis, confirming that the test item was only partially soluble in Daphnia medium and that the concentrations of the test item were satisfactorily maintained over the whole 48 h test period. The effective concentrations ErCx/EyCx were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

Observations

After 24 h of exposure to the test item, following immobilization rates were observed:

62.5% at 219 mg/L

50% at 99.8 mg/L

30% at 45.3 mg/L.

No significant effects (≤10% immobilization) were observed at the loading rates of 20.6 and 9.37 mg/L or in the blank controls.

After 48 h of exposure to the test item, following immobilization rates were observed: 87.5% at 219 mg/L, 77.5% at 99.8 mg/L and 55% at 45.3 mg/L.

No significant effects (≤10% immobilization) were observed at the loading rates of 20.6 and 9.37 mg/L or in the blank controls.

Conclusion

The 48 h EC50 value to Daphnia magna is 8.18 mg/L, based on the measured concentrations of the active ingredient. All validity criteria were fulfilled.