Reaction products of tetrazotized 5-amino-2-[(E)-2-(4-amino-2-sulfophenyl)ethenyl]benzenesulfonic acid with 6-amino-4-hydroxynaphthalene-2-sulfonic acid and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid in 2-[bis(2-hydroxyethyl)amino]ethanol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

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

Reason / purpose for cross-reference:

read-across: supporting information

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, MDA salt (non-preserved, filtrated) 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.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across approach should be used for data gap filling of Bayscript Blaukomponente TEA using environmental fate, ecotoxicity and human health toxicity studies of Bayscript Blaukomponente MDA. This approach is justified by structural similarity, i.e. a nearly identical composition, between source and target substance. Since the same method of manufacturing is used for Bayscript Blaukomponente MDA and Bayscript Blaukomponente TEA the composition of these UVCB substances is nearly identical. Bayscript Blaukomponente MDA (source) and Bayscript Blaukomponente TEA (target) exhibit three main organic salt constituents. Structures are mainly differing in the functional groups of the cationic amines MDA and TEA used as counterions.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Bayscript Blaukomponente TEA is a multi-constituent substance, consisting of three main constituents. The cationic counterion of Bayscript Blaukomponente TEA is 2,2'2" nitrilotris[ethanol] (TEA) (CAS no. 105-59-9).
Bayscript Blaukomponente TEA is manufactured in water and is handled and used as an aqueous solution during its lifetime. It is manufactured as a 34 % solution and in the further handling diluted by mixing with other aqueous colourant solutions to give aquatic inks. The solid substance has only been isolated for REACH registration purposes.

Typical concentrations depend on each registrant’s company specific legal entity composition. No impurity relevant for classification and labelling was identified for the composition.

Bayscript Blaukomponente MDA is a multi-constituent substance, consisting of the same three main constituents as Bayscript Blaukomponente TEA. The counterion of Bayscript Blaukomponente MDA is 2,2'-(methylimino)diethanol (MDA) (CAS no. 102-71-6).
Bayscript Blaukomponente MDA is manufactured in water and is handled and used as an aqueous solution during its lifetime. It is manufactured as a 41 % solution and in the further handling diluted by mixing with other aqueous colourant solutions to give aquatic inks. The aqueous solution has been registered under REACH since it was not possible at the time of registration to extract the solid from the aqueous solution without decomposition.

The concentration ranges for each of the three constituents calculated for the water-free source substance are shown in chapter AE A.2. Typical concentrations depend on each registrant’s company specific legal entity composition. No impurity relevant for classification and labelling was identified for the composition.


3. ANALOGUE APPROACH JUSTIFICATION
The UVCB substances Bayscript Blaukomponente MDA (source) and Bayscript Blaukomponente TEA (target) mainly consist each of three identical organic constituents. The structural formula of these constituents is an estimation as acid form due to the analytical results, other isomers with same molecular weight are possible. At the end of the production process the acid form is neutralized in aqueous solution to an ammonium salt with 2,2'-(methylimino)diethanol (for Bayscript Blaukomponente MDA) and 2,2'2" nitrilotris[ethanol] (for Bayscript Blaukomponente TEA), respectively. The constituents of source and target mainly differ in 2,2'-(methylimino)diethanol (MDA) (CAS no. 102-71-6) and 2,2'2" nitrilotris[ethanol] (TEA) (CAS no.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

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, MDA salt (non-preserved, filtrated) 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

The following results provide read-across data from Bayscript Blaukomonente MDA. The read-across approach is based on high structural similarity between Bayscript Blaukomponente MDA (source) and Bayscript Blaukomponente TEA (target).

A study was performed to assess the toxicity of BAYSCRIPT Blaukomponente, MDA salt (non-preserved, filtrated) 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.

Key value for chemical safety assessment

EC50 for microorganisms:

860 mg/L

Additional information

result should read > 860 mg/L