Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

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 (Carbon and Ammonium Oxidation))

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.

Vehicle:

no

Details on test solutions:

Pre-treatment of test item without ATU
- direct weighings were prepared to give the limit test item concentration of 100 mg/L active ingredient, which equals to 129 mg/L test item
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours
- pH was measured and ranged between pH 5.8 – 6.0
- pH was adjusted to pH 7.1 – 7.2 with NaOH

Pre-treatment of reference compound without ATU
- dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water
- pH was adjusted to pH 7 - 8 with HCl

Pre-treatment of test item with ATU
- direct weighings were prepared to give the different test item concentrations
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours
- pH was measured and ranged between pH 6.0 – 6.1
- pH was adjusted to pH 7.2 – 7.3 with NaOH.
- for the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre; 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test organism and synthetic sewage feed
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Date of collection: 2017-11-13
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 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: 7.8
- Synthetic sewage feed: A synthetic waste water feed was prepared
- pH of the synthetic sewage feed: 7.5 ± 0.5

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

18.2 - 19.3

pH:

8.3 - 8.5
physico-chemical oxygen consumption control: 7.5

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

Exposure conditions
- Test item concentration/s: 100 mg/L (active ingredient), 3 replicates; 100 mg/L (active ingredient) with ATU, 2 replicates
- Control: 6 replicates; 4 replicates with ATU
- Test item concentration in physico-chemical oxygen consumption control: 100 mg/L (active ingredient)
- Concentration of reference compound 3,5-Dichlorophenol: 2.5, 5, 10, 20 and 40 mg/L
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: 17 hours
- Incubation time: 3 hours with permanent aeration

Pre-treatment of test item without ATU
- direct weighings were prepared to give the limit test item concentration of 100 mg/L active ingredient, which equals to 129 mg/L test item
- The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours
- pH was measured and ranged between pH 5.8 – 6.0
- pH was adjusted to pH 7.1 – 7.2 with NaOH.

Pre-treatment of reference compound without ATU
- a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water
- pH was adjusted to pH 7 - 8 with HCl

Pre-treatment of test item with ATU
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours
- pH was measured and ranged between pH 6.0 – 6.1
- pH was adjusted to pH 7.2 – 7.3 with NaOH
- for the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre; 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Details on results:

The effect value relates to a nominal concentration, since no analytical monitoring was performed.
Bayscript Magenta BB showed 0.76 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L active ingredient.
As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L active ingredient no statistical analysis was required to determine the EC50.
The No Observed Effect Concentration was calculated according to STUDENT-t test for Homogeneous Variances using the statistics programme ToxRatPro Version 2.10

Results with reference substance (positive control):

The EC50 of the reference compound 3,5-Dichlorophenol was 16.599 mg/L.

Validity criteria fulfilled:

yes

Remarks:

oxygen uptake rate of blank controls >= 20 mg O2/g of activated sludge in an hour; CV of oxygen uptake in the control replicates <= 30 % at end of test; EC50 of reference compound 3,5-Dichlorophenol 2 – 25 mg/L for total respiration

Conclusions:

Bayscript Magenta BB showed 0.76 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L active ingredient. The EC50 is higher than 100 mg/L (active ingredient). The NOEC is equal or higher than 100 mg/L (active ingredient). The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Executive summary:

A study was performed to assess the toxicity of Bayscript Magenta BB to bacteria. The study was conducted in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02). This test method is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008).

The activated sludge was exposed to Bayscript Magenta BB at a limit test item concentration of 100 mg/L active ingredient. The respiration rate of each mixture was determined after aeration periods of 3 hours.

Six controls without the test item were included in the test design, three at the start and the others at the end of the test series.

A limit test was performed with 3 replicates with a test item concentration of 100 mg/L active ingredient. Each batch of activated sludge was checked using 5 concentrations in the range of 2.5 – 40 mg/L of 3,5-Dichlorophenol as a reference compound. A physico-chemical oxygen consumption control was carried out additionally.

The oxygen uptake rates of four additional controls and two replicates of the test item concentration 100 mg/L active ingredient, all in the presence of N-allylthiourea, were prepared and measured after the exposure period. These values represent the heterotrophic respiration.

Bayscript Magenta BB showed 0.76 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L active ingredient.

The EC50 is higher than 100 mg/L (active ingredient). The NOEC is equal or higher than 100 mg/L (active ingredient).

The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Description of key information

The study was conducted in accordance with OECD Guideline 209. Bayscript Magenta BB showed 0.76 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L active ingredient after 3 h. The EC50 is higher than 100 mg/L (active ingredient). The NOEC is equal or higher than 100 mg/L (active ingredient). The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Should read: EC 50 > 100 mg/L, NOEC >= 100 mg/L.