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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
according to guideline
other: German standard method "Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Pruefung der Atmungshemmung"
equivalent or similar to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
not applicable
GLP compliance:
Analytical monitoring:
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Type: activated sludge
- Origin: laboratory scale sewage treatment unit (3L) treating predominantly municipal sewage. (Location: Southern Wupperverband/Germany)
- no pretreatment
- pH: 7.3
- biomass concentration: 480.0 mg/L
Test type:
Water media type:
Limit test:
Total exposure duration:
30 min
no data
Test temperature:
7.1 - 7.2
Dissolved oxygen:
O2 - Start: 5.0-5.9 mg/L
O2 - End: 2.7-3.2 mg/L
no data
Nominal and measured concentrations:
100, 1000, 10000 mg/L (nominal concentration)
Details on test conditions:
- Test item concentration/s: 100, 1000 and 10000 mg/L
- Test item concentration in physico-chemical oxygen consumption control: 10000 mg/L
- Concentration of reference compound : 5, 10 and 20 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: 480 mg/L dry weight
- Test temperature: 20 ± 2°C
- Incubation time: 30 minutes with permanent aeration
Reference substance (positive control):
3,5-Dichlorophenol (Purity 97 %)
Key result
30 min
Dose descriptor:
Effect conc.:
> 10 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The concentrations of the test item and reference compound were not confirmed by analytical methods, but instead based on nominal concentrations.
Results with reference substance (positive control):
For 3,5-Dichlorophenol an EC50 of 9.2 mg/L has been determined.
The EC50 value was calculated, using Probit–analysis, from the inhibition of the respiration rates at different concentrations.
Validity criteria fulfilled:
on the basic of graphic evaluation: the EC50 for 3,5-DCP was 8.8 mg/L and between the prescribed limits of 2 and 25 mg/L
Solvent Violet 36 showed 7.4 % respiration inhibition of activated sludge at the highest test item concentration (10000 mg/L) and an EC50 of higher than 10000 mg/L was determined. Test concentrations are given as nominal concentrations.
Executive summary:

A study with activated sludge was performed according to the German standard method "Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Prüfung der Atmungshemmung", which is equivalent to the OECD Guideline 209. Two controls without test item were included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge was checked using 3,5-Dichlorophenol as a reference compound. The physico-chemical oxygen consumption control should be carried out, since some substances may consume oxygen by chemical reactivity. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was additionally tested without activated sludge. To measure the oxygen consumption, 250 mL of sludge with test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and O2-content was measured with an O2-meter (redox electrode) with writer. The respiration rate for each concentration was determined graphically from the linear part of the curve of O2-content versus time. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean of the respiration rates of two controls. The sludge was exposed to three different nominal concentrations (100, 1000, 10000 mg/L) of Solvent Violet 36.

Because of the strong oxygen consumption of the activated sludge, only 0.48 g/L was used in all batches. The respiration rate of each mixture was determined after aeration period of 30 min.

Solvent Violet 36 showed 7.4 % respiration inhibition of activated sludge at the highest test item concentration of 10000 mg/L. An EC50 >= 10000 mg/L was determined. Test concentrations are given as nominal concentrations and were not confirmed by analytical methods. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microorganisms.

Description of key information

30min-EC50 (activated sludge) > 10000 mg/L (highest tested concentration); Equivalent to OECD TG 209; Mueller (1997)

Key value for chemical safety assessment

Additional information

Similar results were obtained in the experimental studies on the toxicity to microorganisms on other substances of the category: Reinblau RLW (CAS No 41611-76-1) 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