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Toxicity to microorganisms

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toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Mai - 27 Jun 2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data based on the inhibition control of a ready biodegradability study. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2012).
Reason / purpose for cross-reference:
reference to same study
according to guideline
other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
other: EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Test organisms (species):
other: activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sewage treatment plant, Edenkoben
- Pretreatment: the sludge was filtrated, washed with twice tap water, then washed with and re-suspended in test medium, aerated for ≥ 12 h. The dry matter was determined with 4920 mg suspended solids/L.
- Concentration of sludge: 25 mg dw/L
Test type:
Water media type:
Limit test:
Total exposure duration:
29 d
Details on test conditions:
- Composition of medium: according to guideline
- Test temperature: 22 ± 2 °C
- pH: 6. 76 - 7.47
- Aeration: flasks (with medium and inoculum) were aerated for 24 h with purified, CO2-free, moistened air to purge the system of CO2

- Culturing apparatus: 2000 mL Schott-flasks as test vessels, 100 mL scrubber flasks as absorbent vessels (glassware cleaned with the cleaning agent Mucasol® and then rinsed with tap water, diluted HCL, tap water and deionised water)
- Measuring equipment: IC measurement was conducted using the carbon analyser TOC multi N/O 2100S, Analytik Jena
- Test performed in closed vessels: yes
- Details of trap for CO2: emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask

- Sampling method and frequency: from each front scrubber flask, 9 samples (sample volume 1 mL) were taken in order to determine the emitted CO2 (on days 0, 4, 6, 10, 12, 14, 18, 24 and 29). The resulting change in the volume of the front flask was considered in the calculation of emitted CO2. On day 28, 5 mL 2 M HCl were added to each test flask in order to drive off dissolved CO2.

- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1
- Positive control (with reference substance): 2
Reference substance (positive control):
14 d
Dose descriptor:
Effect conc.:
>= 36.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: Result of toxicity control from ready biodegradability test
Results with reference substance (positive control):
69% degradation of the reference substance after 10 d.

Degradation in the toxicity control was over 25% after 14 d (49%). Therefore, the test item can be considered as not toxic towards the inoculum under tested conditions.

Validity criteria fulfilled:

Description of key information

NOEC (14 d) ≥ 36.1 mg/L (nominal, OECD 301 B)

Key value for chemical safety assessment

Additional information

A toxicity control was performed in the study testing the ready biodegradability of the substance. These results were used to assess the endpoint `toxicity to microorganisms´. A treatment containing the test item at a concentration of 36.1 mg/L and aniline, as a reference substance, at 24 mg/L (nominal), was inoculated with sewage sludge microorganisms and incubated for 28 d. 49% biodegradation was noted within 14 d based on CO2 evolution. A substance can be assumed to be not inhibitory to aquatic microorganisms, if in the toxicity control of a ready biodegradation test, more than 25% degradation based on CO2 evolution occurred within 14 days. Thus, the test item can be assumed to be not inhibitory up to the tested concentration to the activated sludge microorganisms (NOEC (14 d) ≥36.1 mg/L).