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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-01 to 2018-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Appropriate amounts of test item were directly weight into the test vessels and test water was added. The composition was stirred intensively
- Controls: pure water, synthetic sewage and inoculum, but without addition of test item
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test System: Activated sludge
Origin: Municipal sewage treatment plant of Bensheim, Germany
Reason for the selection: Non-adapted activated sludge from the sewage plant at Bensheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
Pretreatment: The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding
prior to use the sludge was fed with 50 mL synthetic sewage per litre and kept aerated at room temperature overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.4°C
pH:
control: 6.5 (start) to 7.5 (end)
test item (6 replicates): 6.5 - 6.7 (start) to 7.2 - 7.4 (end)
Dissolved oxygen:
Control samples : 6.9 mg O2/L
Nominal and measured concentrations:
test item: 10, 32, 100, 320, and 1000 mg/L
positive control: 1, 4 and 16 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks (approx. 1 litre) and Karlsruher flasks (250 mL)
- Type: open
- Aeration:compressed air (1.017 litre per minute)
- No. of vessels per test item concentration (replicates): 5 for total respiration, 5 for heterotrophic respiration (without nitrification)
- No. of vessels per positive control concentration (replicates): 5 for total respiration, 5 for heterotrophic respiration (without nitrification)
- No. of vessels of control (replicates): 6 for total respiration, 6 for heterotrophic respiration (without nitrification)
- Sludge concentration (weight of dry solids per volume): 3.00 g/L
- Nutrients provided for bacteria: no
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Test item concentrations: 10, 32, 100, 320, and 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
3,5-Dichlorophenol
EC50 = 5.9 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The test item had no inhibiting effects above 11% for test item concentrations up to and including 1000 mg/L for total and nitrification respiration. For heterotrophic respiration, significant inhibiting effects on the respiration of activated sludge microorganisms were observed at test item concentrations of 320 and 1000 mg/L.
The NOEC was determined to be ≥ 1000 mg /L for total respiration was determined to be at a test item concentration of 100 mg/L for heterotrophic respiration and was determined to be ≥ 1000 mg/L for nitrification respiration.
The 3-hour EC50 could not be established for total and nitrification respiration and was found to be at a test item concentration of 1552.5 mg/L for heterotrophic respiration.
Executive summary:

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted July 22, 2010.

The test item had no inhibiting effects above 11% for test item concentrations up to and including 1000 mg/L for total and nitrification respiration. For heterotrophic respiration, significant inhibiting effects on the respiration of activated sludge microorganisms were observed at test item concentrations of 320 and 1000 mg/L.

The NOEC was determined to be ≥ 1000 mg /L for total respiration was determined to be at a test item concentration of 100 mg/L for heterotrophic respiration and was determined to be ≥ 1000 mg/L for nitrification respiration.

The 3-hour EC50 could not be established for total and nitrification respiration and was found to be at a test item concentration of 1552.5 mg/L for heterotrophic respiration.

The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration, heterotrophic respiration and for the oxygen uptake due to nitrification was in the range recommended by the test guidelines and confirms the suitability of the activated sludge used.

Description of key information

The test item had no inhibiting effects above 11% for test item concentrations up to and including 1000 mg/L for total and nitrification respiration. For heterotrophic respiration, significant inhibiting effects on the respiration of activated sludge microorganisms were observed at test item concentrations of 320 and 1000 mg/L.

The NOEC was determined to be ≥ 1000 mg /L for total respiration was determined to be at a test item concentration of 100 mg/L for heterotrophic respiration and was determined to be ≥ 1000 mg/L for nitrification respiration.

The 3-hour EC50 could not be established for total and nitrification respiration and was found to be at a test item concentration of 1552.5 mg/L for heterotrophic respiration.

Key value for chemical safety assessment

EC50 for microorganisms:
1 552.5 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information