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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8.10. – 17.10. 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
(see Overall remarks)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
The activated biological sludge containing mixed culture of microorganisms obtained from the second step of sewage treatment plant of Pardubice was used for the testing. The wastewater processed by the sewage treatment plant is predominantly municipal.

PREPARATION OF THE INOCULUM
The activated sludge was collected two days before the day of testing. After the sample collection the sludge was washed with potable water for 0.5 hour and subsequently decanted for 0.5 hour. This procedure was repeated three times in total.
Further the sludge was modified by addition of 50 mL of cultivation medium per 2 L of diluted sludge suspension at permanent aeration till the day of test.
The dry weight was determined from 10 mL of sludge suspension after 0.5 hour sedimentation.
Before the test the sludge was suspended in water up to concentration about 4 000 mg of sludge dry weight per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.

JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM
The activated sludge used in the test is in conformity with the recommendations of the test guidelines.
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
pH:
approx. 6 – 8
Nominal and measured concentrations:
100, 180, 320, 560 and 1000 mg.L-1 (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: oxygen vessels (BOD bottles) with volume of about 280 mL for measurement of oxygen concentration
- Type (delete if not applicable): closed
- Aeration: by filtered pressured air
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge dose on concentration of 4000 mg.L-1 : 303 mL.L-1

WATER PARAMETERS
- deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: was not carried out
- Photoperiod: daily lighting

TEST PROCEDURE
Defined dose of the test substance or reference substance , 100 mL of deionized water, 16 mL of the synthetic nutrient medium and 200 mL of the prepared sludge suspension were introduced into each test container. The volume was replenished up to 500 mL with deionized water.
The control samples were prepared only from the same volume of nutrient medium and sludge and replenished up to 500 mL with water.
The check of abiotic decomposition was performed at the highest concentration of the test substance and nutrient medium without sludge and with addition of 1 mL of mercury chloride solution.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The decline of oxygen concentration was measured and recorded at minimal 10-minute intervals (respiration rate of activated sludge)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8 (100, 180, 320, 560 and 1000 mg.L-1 )
- Range finding study
- Test concentrations: 46, 100, 220, 460 and 1 000 mg.L-1 (spacing factor 2.2)
- The preliminary test was performed to determine the test substance concentrations to be used in the main test.
Reference substance (positive control):
yes
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 8.2 mg.L-1 (95% confidence interval: 6.2 – 9.9 mg.L-1)
Validity criteria fulfilled:
yes
Conclusions:
On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Solvent Yellow 124, the EC50 was found to be:
EC50 > 1 000 mg.L-1
Executive summary:

The influence of the test substance, Solvent Yellow 124, on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

Test was performed according to method C.11 - Activated Sludge Respiration Inhibition Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

The preliminary test was performed using 5 concentrations in range 46 – 1 000 mg.L-1 with a geometric factor of 2.2 to determine the concentrations to be used in the main test.

In the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 (100, 180, 320, 560, 1000 mg.L-1) were used together with two control experiments (without the test substance), one at the beginning and one at the end of the test.

3,5-dichlorophenol was used as reference substance. Five concentrations in geometric progression with a factor of 1.4 (5.2, 7.3, 10, 14 and 20 mg.L-1) were selected.

The abiotic decomposition was not detected even at the highest concentration of the test substance.

The pH value during the main test was in the range of 7.0 to 7.8.

Results of the respiration inhibition test

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Solvent Yellow 124, the EC50 was found to be:

EC50 > 1 000 mg.L-1

Under the current experimental conditions, all tested concentrations in the main test caused no significant inhibition of respiration rate.Therefore it was not possible to calculate the EC50 value.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Solvent Yellow 124, the EC50 was found to be:

EC50 > 1 000 mg.L-1