Sodium 2,4-diamino-5-(2-hydroxy-5-nitrophenylazo)benzenesulphonate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From the 4th to the 14th of October, 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Mordant Brown 033

IUPAC Name:

Mordant Brown 033

Test material form:

other: solid

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
To prepare the standard solutions, a stock solution (test item dissolved in water:methanol (1:1, v:v)) was diluted with Daphnia medium:methanol (9:1, v:v) containing 10 mM ammonium acetate.
Test samples were diluted with methanol containing 100 mM ammonium acetate to obtain a final sample composition of sample:methanol (9:1, v:v) containing 10 mM ammonium acetate before HPLC analysis. If necessary, the samples were further diluted into the calibration range using Daphnia medium:methanol (9:1, v:v) containing 10 mM ammonium acetate.

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: continuosly areated 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:

20.8 - 21 °C
Determined in an additional flask without Daphnia at the beginning of the test and after 24 and 48 h of exposure.

pH:

6.5 - 7.1
Determined in the combined test solutions for each concentration at the beginning and after 48 h of exposure.

Dissolved oxygen:

6.4 - 7.4 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 (>3.0 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:

659 - 685 µS.cm-1
Determined in the combined test solutions for each concentration at the beginning of the exposure.

Nominal and measured concentrations:

Nominal concentration 134 mg/L (100 mg/L active ingredient).

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


.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

PRELIMINARY, NON-GLP TEST
The HPLC measurements indicated that the test item is stable.
% immobile Daphnia after 48 h of exposure: 0% at 100 mg/L nominal concentration.
DEFINITIVE TEST
No significant effects (≤10% immobilization) were observed at 100 mg/l nominal concentration or in the blank controls, neither after 24 h of exposure nor after 48 h.
Therefore, the median effect concentrations (EC50) on Daphnia magna after 24 h as well as after 48 h of exposure were estimated to be >100 mg/l nominal concentration of the active ingredient.
The NOEC values after 24 as well as after 48 h of exposure were both 100 mg/L nominal concentration of the active ingredient.

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

Definition and determination of the effect concentrations (ECx)

The effect concentration ECx is the concentration estimated to immobilise 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 can still move their antennae).

The effect concentration EC50 for 24 and 48 h were estimated based on the concentrations tested and immobilization observed.

Definition and determination of the no observed effect concentration (NOEC)

The no observed effect concentration (NOEC) is the highest test concentration for which the test item is observed to have no biologically significant effects when compared to the 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): > 100 mg/L
EC50 Daphnia magna (48h): > 100 mg/L
NOEC Daphnia magna (48h): > 100 mg/L
nominal concentration of active ingredient

Executive summary:

Method

The median effect concentration (EC50) of to Daphnia magna were investigated under static exposure conditions over a period of 48 h, following guideline OECD 202.

The test item is solid, highly soluble and the test solution was prepared by dissolving the test item directly in Daphnia medium.

The single investigated nominal concentration was 134 mg/L corresponding to 100 mg/L active ingredient. Therefore, this test represents a limit test. 40 individual Daphnia divided into 2 test vessels were exposed to each concentration of the test item, and 60 Daphnia divided into 3 test vessels were used as blank controls.

The concentration in the test medium was measured by HPLC analysis at the beginning and after 24 and 48 h of exposure.

Observations

the test item was completely dissolved and that the concentration of the test item was stable over the whole 48 h period and was within 80-120% of the nominal concentration. Therefore, the effective concentrations ECx were assessed based on the nominal concentration of the active ingredient.

No significant effects (≤10% immobilization) were observed at 100 mg/L nominal concentration or in the blank controls, neither after 24 h of exposure nor after 48 h.

Conclusion

The median effect concentrations (EC50) on Daphnia magna after 24 h as well as after 48 h of exposure were estimated to be >100 mg/L nominal concentration of the active ingredient. All validity criteria were fulfilled.

The NOEC values after 24 as well as after 48 h of exposure were both 100 mg/L nominal concentration of the active ingredient.