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Ecotoxicological information

Toxicity to microorganisms

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toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jul - 26 Sep 2017
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 OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Temperature range was 19.4 – 21.6 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range, this is considered as uncritical concerning the outcome of the study.
according to guideline
other: EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Temperature range was 19.4 – 21.6 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range, this is considered as uncritical concerning the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
Details on test solutions:
- Method: The amounts of test item (28.8 mg/L) and reference item (26.8 mg/L) were directly weighed into the test flasks.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: Sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf, Germany; Date of collection: 25. Aug. 2017
- Preparation of inoculum for exposure: inoculum was taken from its source,washed, aerated and the dry matter was determined; test vessels were filled with medium and inoculum and aerated afterwards.
- Pretreatment: sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use for 96 hours with purified, CO2-free, moistened air to purge the system of CO2
- Concentration of sludge: 4120 mg suspended solids/L
- Inoculum concentration: 25.0 mg/L dry matter/L
Test type:
Water media type:
Limit test:
Total exposure duration:
14 d
Details on test conditions:
- Composition of medium: according to the OECD guideline 301
- Test temperature: 19.4 – 21.6 °C (see deviation in section „Any other information on material and methods incl. tables“)
- Suspended solids concentration: dry matter was determined as 4600 mg suspended solids/L
- Continuous darkness: incubated in the dark

- Culturing apparatus: 1000 mL-SCHOTT-flasks were used as test vessels, 100 mL scrubber flasks as absorbent vessels.
- Number of culture flasks/concentration: 1 reactor per toxicity control
- Method used to create aerobic conditions: The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels. Magnetic stirrers were used to prevent deposition of inoculum.
- Measuring equipment: Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) every month. After every start, quality control samples were measured.
- Details of trap for CO2 and volatile organics if used: The 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 frequency: On the 0, 2nd, 4th, 7th, 9th, 11th, 14th, 18th, 23trd and 29 day
- Sampling method: From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2. The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2. On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.
Reference substance (positive control):
14 d
Dose descriptor:
Effect conc.:
>= 28.8 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):
Test item is not inhibitory to aerobic activated sludge microorganisms since degradation was >25% within 14 days (80% after 14 d).

The toxicity control attained 80% degradation after 14 days of incubation.

If in a toxicity test, containing both the test and reference substance, less than 35% degradation (based on total DOC) or less than 25% (based on total ThOD or ThCO2) occurred within 14 days, the test substance can be assumed to be inhibitory.” (OECD guideline 301)

Since more than 80% degradation occurred in the toxicity control, the substance is with high probability not toxic to aquatic microorganisms. The test item concentration in the toxicity control of 28.8 mg/L can be regarded as NOEC value for the toxicity to aquatic microorganisms.

Validity criteria fulfilled:

Description of key information

NOEC (14 d) >= 28.8 mg/L (nominal, toxicity control, OECD 301B, activated sludge)

Key value for chemical safety assessment

Additional information

Since no studies on the toxicity to aquatic microorganisms are available the results of a ready biodegradability study are used to draw conclusion on the toxicity of the substance to aquatic microorganisms. If a compound biodegrades readily in a biodegradability test or does not inhibit at a certain concentration the degradation of a reference substance in the positive control the substance is not considered toxic to microorganisms and the concentration applied in the toxicity control can be set as the NOEC value. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2017). 

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 ThOD occurred within 14 days (OECD guideline 301B).

For the test item a biodegradation test according to OECD guideline 301B is available. The test includes a toxicity control, which contains 28.8 mg/L of the test item and 26.8 mg/L aniline. The toxicity control attained 80.3 % degradation after 14 days of incubation. Hence, the substance is considered not toxic to aquatic microorganisms and the test item concentration of 28.8 mg/L can be used as NOEC.