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Toxicity to microorganisms

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toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
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 301F
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Details on test solutions:
- Method: The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control and functional control) or were prepared directly in brown glass bottles (test item and toxicity control).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, Hildesheim, Germany
- Storage conditions: aerobic (aeration with CO2-free air)
- Storage length: 6 d
- Preparation of inoculum for exposure: aqueous phase of non-adapted activated sludge
- Pretreatment: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in aerobic conditions by aeration during 2.5 h. Thereafter, the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 6 d.
- Initial cell/biomass concentration of the inoculum: 1.18E+09 colony forming units/L (standard dilution plate count)
Test type:
Water media type:
Limit test:
Total exposure duration:
14 d
Details on test conditions:
- Composition of medium: Mineral salts medium according to OECD 301 F.
- Test temperature: 20.5 - 21.1°C
- pH: 7.68 - 7.78 (at test start), 7.66 - 8.07 (at test end)
- Colony forming units (CFU) in the test vessels: approximately 1.18E+07 CFU/L
- Continuous darkness: Yes, in an incubator.
- Other: Continuous stirring

- Culturing apparatus: brown glass bottles (500 mL)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration with CO2-free air.
- Measuring equipment: OxiTop measuring heads, WTW
- Test performed in open system: No, the bottles were closed with OxiTop measuring heads.
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the bottles to absorb evolved CO2.
- Other: Test volume: 250 mL

- Sampling frequency: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system every 112 min during the 28 d incubation period (360 measuring points).

- Inoculum blank: 2 replicates containing mineral medium stock solutions, ultrapure water and inoculum.
- Functional control: 1 replicate containing reference item (45 mg/L = 75.2 mg O2/L) dissolved in mineral medium stock solutions, ultrapure water and inoculum.
- Toxicity control: 1 replicate containing the test item and reference item dissolved in mineral medium stock solutions, ultrapure water and inoculum.
- Other: Separate replicates of the test item and toxicity control were prepared for measurement of the pH-value at test start.
Reference substance (positive control):
Sodium benzoate
Key result
14 d
Dose descriptor:
Effect conc.:
38 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):
The functional control (sodium benzoate + inoculum) reached the pass level for ready biodegradation (> 60% degradation) on Day 3. The validity criterion that the degradation should be > 60% after 14 d was fulfilled.
Stages of Biodegradation: The adaptation phase changed to degradation phase within 2 d (degradation > 10%) and the biodegradation reached a maximum of 92% on Day 23.

In the toxicity control the biodegradation achieved 84% after 14 d. After 28 d the biodegradation reached 92%. The validity criterion that the test item should not inhibit the biodegradation of the reference item (sodium benzoate) was fulfilled.

"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 d, the test substance can be assumed to be inhibitory." (OECD guideline 301).

Since more than 25% degradation occurred after 14 d (and 28 d) incubation of the reference substance with the test substance (toxicity control), it is highly likely that the test substance is not toxic to aquatic microorganisms. The nominal test item concentration of 38 mg/L used in the toxicity control can be used as NOEC value for the toxicity to aquatic microorganisms.

Description of key information

NOEC (14 d) = 38 mg/L (nominal, OECD 301 F)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
38 mg/L

Additional information

Since no studies on the toxicity to aquatic microorganisms are available a ready biodegradability study is used to derive a NOEC for the toxicity to aquatic microorganisms. If a compound degrades well in a ready biodegradability test, or does not inhibit the degradation of a positive control at a certain concentration, this concentration can be used as a NOEC value. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2008).

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 oxygen demand (BOD/ThOD) occurred within 14 days (OECD guideline 301). For the present substance a biodegradation test according to OECD guideline 301 F is available. The test includes a toxicity control, which contains 38 mg/L of the test item and 45 mg/L of the reference material (sodium benzoate). The toxicity control attained 84% and 92% degradation after 14 d and 28 d of incubation, respectively. Hence, the substance is not toxic to aquatic microorganisms in the toxicity control and the test item concentration of 38 mg/L can be used as NOEC.