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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:
16 - 17 Feb 2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
The rate of respiration in the 100 mg/L test vessel was > than that in the replicate control vessels and could not be measured within the linear portion of the respiration curve as the start of the linear portion of the trace was lower than 1.5 mg O2/L.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on test solutions:
Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 and 8.0.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK. The plant treats predominantly domestic sewage.
- Initial biomass concentration: suspended solids concentrations was equal to 3.0 g/L
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
prior to adjustment: 5.7 - 6.0
After adjustment: 7.3 - 7.9
Nominal and measured concentrations:
Nominal test substance concentrations: 10, 100 and 1000 mg/L
Details on test conditions:
- Test vessel: conical flask
- Fill volume: 500 mL
- No. of vessels per concentration (replicates): 1 resplicate of 10 and 100 mg/L test concentration respectively; 3 replicates of 1000 mg/L test concentration
- No. of vessels per control (replicates): 4
- No. of vessels per reference control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L
- Nutrients provided for bacteria: synthetic sewage

- Source/preparation of dilution water: deionized reverse osmosis water containing < 1 mg/L DOC.

Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
The reference item gave a 3-Hour EC50 value of 7.9 mg/L, 95% confidence limits 6.2 - 10 mg/L.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Description of key information

The substance is not toxic to microorganisms.

Key value for chemical safety assessment

Additional information

The toxicity of the substance to microorganisms was tested in a guideline study according to OECD 209. Activated sludge was exposed to test substance concentrations of 10, 100 and 1000 mg/L for 3 hours. The respiration of the inoculum was not affected. An EC50 >1000 mg/L was determined.

Hexanoic acid, 2-ethyl-, C16-18 alkyl esters (CAS No. 90411-68-0) is not toxic to microorganisms.