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Administrative data

activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Comparable to guideline study Hypothesis and analogue approach for read-across: This substance (S-Ethanol, composition 2) has degree of ethanol purity between 76 - 82%. Methanol is the main impurity of the target substance (conc. 13-14 %), and considered the major driver for adverse effects based on its properties and relative quantity in the substance. Therefore, testing of the substance itself was considered unnecessary. Instead the available REACH registration data of ethanol and methanol is used in order to update the chemical safety assessment of this substance. Other impurities of this substance are taken into account for self-classification, but there is no need to consider their properties in hazard assessment because of low concentrations (see IUCLID section 1.2; composition 2).

Data source

Reference Type:
Evaluation of the OECD activated sludge, respiration inhibition test
Klecka, G.M. et al.
Bibliographic source:
Chemosphere 14(9): 1239-1251

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Automatically generated during migration to IUCLID 6, no data available
Automatically generated during migration to IUCLID 6, no data available
Details on test material:
- Name of test material (as cited in study report): Methanol
- Analytical purity: reagent grade

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: a portion of a reaction mixture was transferred to a standard BOD dilution bottle.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions (0.5 to 5.0 g/L) of the test chemical were prepared in deionized water. When necessary, the stock solutions were adjusted to pH 7.5 ± 0.5 by the addition of 1N H2SO4 or 1N NaOH as required

Test organisms

Test organisms (species):
other: activated sludge from domestic and industrial sewage treatment plants
Details on inoculum:
Activated sludge was obtained on the day prior to the first day of each series of inhibition tests. On return to the laboratory, the solids were allowed to settle and the waste liquor was discarded. The solids were then transferred into a 9-liter laboratory-scale, semi-continuous activated sludge cylinder and diluted to approximately 8.5 liters with deionized water. The system was initially mixed by aerating at a rate of approximately 0.5 L/min, and the concentration of mixed liquor suspended solids was determined by gravimetric analysis. The solids were then allowed to settle in the cylinder and the upper layer of waste liquor was discarded. The activated sludge was washed three times with appropriate volumes of deionized water. After washing, the system was adjusted to contain 4000 ± 400 mg of mixed liquor suspended solids (dry weight) per liter. The system was then aerated continuously at a rate of 0.5 L/min and incubated at ambient temperature (21°C).
The system was supplemented daily with 50 mL of a synthetic sewage stock solution per litter of activated sludge. The synthetic sewage stock solution was composed of, per liter: Bacto-Peptone, 16.0 g; Bacto-Beef extract, 11.0 g; urea 3.0 g; K2HPO4, 28 g; MgSO4 • 7H2O, 0.2 g; CaCl2 • 2H2O, 0.4 g; and NaCl, 0.7 g. Final pH of the stock solution was adjusted to pH 7.0 with H3PO4. Fresh stock solution was prepared as required and stored for not more than two days at 5°C.
When the same source of inoculum was to be used for testing on a subsequent day (maximum of 7 days). an additional 50 mL of synthetic sewage stock solution was added per liter of activated sludge. The system was incubated overnight.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h

Test conditions

Test temperature:
7.4 - 8.0
Details on test conditions:
- Test vessel:
- Material, size, headspace, fill volume: 1L-bottle, final volume 500 mL
- Aeration: 0.5 - 1.0 L/min

- Source/preparation of dilution water: deionized water

- Adjustment of pH: yes, 7.5 ± 0.5
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
During a number of preliminary inhibition studies, it was observed that the pH of a test reaction often changed during the 3-hour incubation period. Thus, decreases in the respiration rate may have been due to a combination of both the effects of pH and toxicity of the test chemical. As a consequence, the buffering capacity of the synthetic sewage nutrient medium was increased to prevent changes in pH of reaction mixtures.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: IC50 for 3,5-dichlorphenol: 12.2 ± 2.2 mg/L (18% variance) (municipal sewage), 11.4 ± 1.5 mg/L (13% variance) (industrial sewage)
Reported statistics and error estimates:
Inhibition data were analyzed using Thompson's method of moving averages to estimate IC50 values. The experimental data were also analyzed using a probit-transformation model similar to that described by Larson and Schaeffer (1982). A nonlinear curve-fitting program (Procedure NLIN of SAS) was used to estimate IC50 values and associated 95% confidence intervals.

Applicant's summary and conclusion