1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

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

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 4000 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 volume with deionised
water.
- Storage of sludge : aeration of the activated sludge at 20°C ± 2°C, daily fed with synthetic medium
- pH of the suspension before application : 7.4

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

No data

Test temperature:

20.4 °C (concerning the highest test concentration)

pH:

8.1 (concerning the highest test concentration)

Dissolved oxygen:

- At the beginning: 6.2 mg/l (concerning the highest test concentration)
- At the end: 2.8 mg/l (concerning the highest test concentration)

Salinity:

No data

Nominal and measured concentrations:

10, 100 and 1000 mg/l (nominal), no analytical monitoring was performed

Details on test conditions:

Pre-treatment of test item and reference compound:
- Direct weighings were prepared to give the different test item concentrations. 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 16 hours.
- For the reference compound 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. The pH was adjusted to pH 7 ± 0.5 with HCl.

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:
- Test vessels : 300 mL glass Erlenmeyer flasks
- Method of application : direct weighing
- Concentration of reference compound 3.5-Dichlorophenol : 2.5, 5, 10, 20 and 40 mg/l
- 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

Reference substance (positive control):

yes

Remarks:

(3,5-dichlorophenol (Acros Organics, Batch number A0298018, purity: 99.6 %)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

10 mg/l: 0 % respiration inhibition
100 mg/l: 0 % respiration inhibition
1000 mg/l: 0 % respiration inhibition

Results with reference substance (positive control):

2.5 mg/l: 17.1 respiration inhibition
5.0 mg/l: 19.4 respiration inhibition
10 mg/l: 50.8 respiration inhibition
20 mg/l: 83.3 respiration inhibition
40 mg/l: 89.2 respiration inhibition

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:

The test item showed 0 % respiration inhibition of activated sludge after a test period of 3 hours at the highest concentration of 1000 mg/L. Therefore an EC50 of higher than 1000 mg/L could be calculated for the test substance. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Executive summary:

In order to test the toxicity to microorganism of the substance, activated sludge from predominantly domestic sewage plant was exposed to the test solution of three nominal concentrations of the substance (10, 100, 1000 mg/l), blank control solution and solution with positive reference substance for a period of 3 hours. Dissolved oxygen was recorded and respiration rates were calculated. 0 % respiration inhibition was observed at the highest concentration of test substance as water solution and therefore an EC50 (3h) < 1000 mg/L. The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Activated sludge respiration inhibition” (2008). This test method is equal to OECD Guideline 209 (1984). This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microorganisms.

Description of key information

3h-EC10 and 3h-EC50 (activated sludge) > 1000 mg/L (highest tested concentration); EU Method C.11; Currenta (2012)

Additional information

Similar results were obtained in the experimental studies on the toxicity to microorganisms on other substances of the category: Solvent Violet 36 (CAS No 82-16-6) and Solvent Blue 104 (CAS No 116-75-6). See the category approach justification document, attached in section 13, for further details.

Global overview of the category approach for the endpoint:

 

	Solvent Violet 36	Solvent Green 3	Reinblau RLW	Reinblau BLW	Solvent Blue 104^#	Solvent Green 28
Toxicity to microorganisms	30min-EC50 (activated sludge) > 10000 mg/L (nominal)^$	Read across to Reinblau RLW   Other Lead Registrant data available but not considered key	3h-EC10 and EC50 (activated sludge) > 1000 mg/L (nominal)^$	Study planned (OECD TG 209)   Read across to Reinblau RLW	3h-EC50 and NOEC (activated sludge) > 1000 mg/L and ³ 1000 mg/L (nominal)^$	Study planned (OECD TG 209)

^# not registered by LANXESS, data consulted on ECHA disseminated website on April, 11 2022

^$ experimental studies