Reaction mass of lithium sodium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxynaphthalene-2,7-disulfonate 2,2'-(methylimino)diethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

direct weighings

Test organisms (species):

activated sludge

Details on inoculum:

- Type : mixed population of aquatic microorganisms (activated sludge)
- Origin : aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
- Date of collection : 2012-08-13
- Microbial inoculum : The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 4500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water. Storage of sludge : aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.5

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

pH:

7.7

Dissolved oxygen:

O2 start: 6.3 mg/L
O2 end: 2.7 mg/L

Nominal and measured concentrations:

Test item concentration/s (active ingredient): 8.74, 86.2 and 860 mg/L (nominal)
Test item concentration in physico-chemical oxygen consumption control: 860 mg/L

Details on test conditions:

Test system:
Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8.
8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C.
Control vessels (inoculated sample without test item) were prepared the same way.
Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge.
Oxygen consumption was measured and recorded after an aeration time of 3 hours in all these vessels starting with control 1. Thereafter, temperature and pH were measured as well. Then the other test vessels were measured. Control 2 terminated the measurements.
Exposure conditions:
The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.

- Test vessels : 300 mL glass Erlenmeyer flasks
- Method of application : direct weighing
- Test concentration of the activated sludge : 800 mg/L suspended solids
- Test temperature : 20 ± 2°C
- Stirring period of the test item before start of incubation time : 16 hours
- Incubation time : 3 hours with permanent aeration

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Purity: 99.6 %). Concentration of reference compound: 2.5, 5, 10, 20 and 40 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 860 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 860 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Concentrations are given as nominal concentrations and were not confirmed by analytical methods.

Validity criteria fulfilled:

yes

Remarks:

respiratory rates of the 2 controls differ less than 15 % from each other; the EC50 of the reference compound 3,5-Dichlorophenol is in the range 5 – 30 mg/L

Conclusions:

A test with activated sludge with a duration fo 3 hours was performed according to the OECD Guideline 209. BAYSCRIPT Blaukomponente (unkonserviert, filtriert) showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 860 mg/L.

Executive summary:

A study was performed to assess the toxicity of BAYSCRIPT Blaukomponente (unkonserviert, filtriert) to bacteria. The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984). The activated sludge was exposed to BAYSCRIPT Blaukomponente (unkonserviert, filtriert) at different concentrations. The respiration rate of each mixture was determined after aeration periods of 3 hours. The substance showed 0.0 % respiration inhibition of activated sludge at a test item concentration of 860 mg/L. The EC50 is higher than 860 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

Description of key information

A test with activated sludge with a duration fo 3 hours was performed according to the OECD Guideline 209. BAYSCRIPT Blaukomponente (unkonserviert, filtriert) showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 860 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

860 mg/L

Additional information

should read > 860 mg/L

In accordance with REACH Regulation, Art. 3, and ECHA "Guidance for identification and naming of substances under REACH and CLP" v1.2, March 2012, substances have to be separated from solvents if this is not affecting the stability of the substance or changing its composition.

In this case, separation of water would only be possible by thermical damaging of the substance. Furthermore, it was not possible to spray dry the pigment solution as it stuck to and partly melted on the conus dryer.