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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 04 - Sep 17, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 Oct 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 Darmstadt, 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 feed (see below) 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. The pH of the activated sludge inoculum was 6.9 and therefore no adjustment necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.4°C
pH:
control: 7.3 (start) to 8.6 (end)
test item: 7.3 (start) to 8.6 (end)
Dissolved oxygen:
Control samples : 6.9 mg O2/L
Nominal and measured concentrations:
test item: 100 mg/L (nominal)
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): 1
- No. of vessels per positive control concentration (replicates): 1
- No. of vessels of control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 3.5 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: 100 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
At a nominal test concentration of 100 mg test item/L less than 10% inhibition was observed after three hours of
incubation. The mean deviation in respiration rate was 4.2% at three replicates of 100 mg of the test item.
The respiration rate of the activated sludge to the test material at the rate of 100 mg/L was not statistically significant reduced when compared to the control (Two
sample t-test procedure). The test material at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge.
Therefore it can be assumed that the NOEC is above 100 mg test item/L.
Results with reference substance (positive control):
3,5-Dichlorophenol
EC50 = 8.1 mg/L
Validity criteria fulfilled:
yes
Conclusions:
At a nominal test concentration of 100 mg test item/L less than 10% inhibition was observed after three hours of incubation. The mean deviation in respiration rate was 4.2% at three replicates of 100 mg test material.
The respiration rate of the activated sludge to the test material at the rate of 100 mg/L was not statistically significant reduced when compared to the control (Two sample t-test procedure). The test material at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. Therefore it can be assumed that the NOEC is above 100 mg test item/L.
Executive summary:

Purpose

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item on the activity of activated sludge by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours.

Study Design

The study was performed according to OECD Guideline 209, Activated Sludge, Respiration and Inhibition Test (Carbon and Ammonium Oxidation), adopted 22 October 2010.

The test item was directly dosed into each test flask and pure water was added. The test item was dissolved into the pure water as homogeneously as possible.

Test Concentrations

100 mg test item (nominal);

1, 4 and 16 mg 3,5 -Dichlorophenol/L (positive control) and

2 inoculum controls.

Results

Toxicity of Test Item

At a nominal test concentration of 100 mg test item/L less than 10% inhibition was observed after three hours of incubation. The mean deviation in respiration rate was 4.2% at three replicates of 100 mg test material

The respiration rate of the activated sludge to the test material at the rate of 100 mg/L was not statistically significant reduced when compared to the control (Two sample t-test procedure). The test material at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. Therefore it can be assumed that the NOEC is above 100 mg test item/L.

Toxicity of 3.5 -Dichlorophenol

The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration was found to be 8.7 mg/L thus lying in the range of 5 - 30 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.

Description of key information

The test item had no inhibiting effects on the respiration rate of activated sludge up to and including 100 mg/L.

The NOEC was determined to be > 100 mg /L. The 3-hour EC10, EC20, and EC50 values could not be established.

Key value for chemical safety assessment

Additional information