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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-30
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:
The temperature in the experiment was in a range of 20.3 – 22.6 °C instead of 20.0 ± 2.0 °C.
This deviation was stated as uncritical, as normal respiration activity of the control could be observed.
Deviations:
yes
Remarks:
please refer to 'version/remarks'
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz (8.04.2015)
Specific details on test material used for the study:
The test item was stored in a closed vessel at room temperature (20 ± 5°C).
Analytical monitoring:
no
Details on sampling:
- Concentrations: 0, 10, 32, 100, 320, 1000 mg/L
- Sampling method: After 3 hours, the content of the first vessel was shaken vigorously for 30 seconds, poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes . The following vessels were measured likewise in 5 minutes intervals
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The effects on micro-organisms from activated sludge were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance.
- Controls: blank control, positive control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The sludge was taken from the activation basin of the sewage treatment plant in D-67480 Edenkoben.
- Method of cultivation: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed/L.
- Preparation of inoculum for exposure: Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution/L sludge. On the day of the experiment, the dry matter was determined once more.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
The respiration rate was measured over a period of only 5 minutes.
Hardness:
0.71
Test temperature:
20.3 – 22.6 °C
pH:
7.7 - 8.1
Conductivity:
174 µS/cm
Nominal and measured concentrations:
nominal: 0, 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: SCHOTT-flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 2000 mL
- Aeration: purified air, using Pasteur pipettes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 1
- No. of vessels per blank control (replicates): 2 replicates before and 2 after measuring positive control and test item, respectively
- Sludge concentration (weight of dry solids per volume): 1.1 g/L in test
- Nutrients provided for bacteria: 16 mL of nutrient solution in 250 mL test solution
Composition of nutrient solution (synthetic sewage):
Peptone 16.0 g
Meat Extract 11.0 g
Urea 3.0 g
NaCl 0.7 g
CaCl2*2H2O 0.4 g
MgSO4*7H2O 0.2 g
K2HPO4 2.8 g
Deionised water ad 1000 mL

- Nitrification inhibitor used: none
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : NOEC, EC10, EC50
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Concentrations were based on a non-GLP range finding test.
- Test concentrations: 0, 10, 32, 100, 320, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (CAS-No. 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L was freshly prepared for the experiment
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % confidence interval 56 - 77 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % confidence interval 220 - 280 mg/L
Details on results:
- Blank controls oxygen uptake rate: 34.28 mg/h/g activated sludge
- Coefficient of variation of oxygen uptake rate in control replicates: 6.9 %
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (3 h) = 13 mg/L (All values lie within the recommended range of 2 – 25 mg/L.)
Reported statistics and error estimates:
Determination of NOEC: For the treatments with the test item concentrations 100 and 32 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used (As equality of variance is given, the t-test is used.)
Determination of EC10, EC5: For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram.
EC10 and EC50 were determined from the x-values of the regression line at y = 10 % and y = 50 %.

One valid experiment according to OECD 209 was performed. In the experiment the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. The concentrations tested were based on a non-GLP range finding test. To avoid volatilization and for safety reasons, the experiment was performed in a closed system without aeration. In order to ensure sufficient oxygen supply, a large headspace was provided and the mixtures were stirred vigorously. This procedure is described in the OECD guideline. All validity criteria were met. For the estimation of the EC50 of the positive control, a good statistical correspondence of the data with the dose response-equation was found. The positive control gave an EC50 of 13 mg/L which lies within the recommended range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 hour. The concentration 32 mg/L of the test item hexanal can be stated as NOEC. The EC10 was determined with 67 mg/L and an EC50 of 250 mg/L was deduced from this study. No inconsistencies in the dose-response estimation could be observed. Therefore, no further experiment was performed in order to discern between inhibition of nitrificators and inhibition of total population. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Validity criteria fulfilled:
yes
Conclusions:
The concentration 32 mg/L of the test item hexanal can be stated as NOEC. The EC10 was determined with 67 mg/L and an EC50 of 250 mg/L was deduced from this study.
Executive summary:

The inhibition of the respiration of activated sludge by hexanal was investigated according to OECD 209 and in compliance with GLP criteria. In the experiment hexanal was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. The concentrations to be tested were based on a non-GLP range finding test. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.2 g suspended solids/L, giving a concentration of 1.1 g suspended solids/L in the test. One valid experiment was performed. 3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 13 mg/L (95 % confidence interval: 7.8 – 25 mg/L) was determined, which lies within the recommendedrange of 2 – 25 mg/L stated in the OECD guideline. The following results for the test item hexanal were determined:

 

3h NOEC = 32 mg/L

3h EC10 = 67 mg/L (confidence interval: 56 – 77 mg/L)

3h EC50 = 250 mg/L (confidence interval: 220 – 280 mg/L)

Description of key information

The inhibition of the respiration of activated sludge by hexanal was investigated according to OECD 209 and in compliance with GLP criteria. In the experiment hexanal was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. The concentrations to be tested were based on a non-GLP range finding test. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.2 g suspended solids/L, giving a concentration of 1.1 g suspended solids/L in the test. One valid experiment was performed.  

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 13 mg/L (95% confidence interval: 7.8 – 25 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline.

 

The following results for the test item hexanal were determined:

 

3h NOEC = 32 mg/L

3h EC10 = 67 mg/L (confidence interval: 56 – 77 mg/L)

3h EC50 = 250 mg/L (confidence interval: 220 – 280 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:
250 mg/L
EC10 or NOEC for microorganisms:
67 mg/L

Additional information