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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-28.11.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is non-soluble in water in concentrations needed for the test. For this reason direct dosing of the test substance was used. The substance was weighed on a slide and immersed into 100 mL of deionised water in the reaction container.
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 activated biological sludge containing a mixed culture of microorganisms was obtained from the second step of the sewage treatment plant of Pardubice. The wastewater processed by the sewage treatment plant is predominantly municipal.
- Preparation of inoculum for exposure: 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 hours and subsequently decanted for 0.5 hours. This procedure was repeated three times in total. 50 mL of cultivation medium was added per 2 L of diluted sludge suspension at permanent aeration till the day of test.
- Pre-treatment: The pH was adjusted to 6.0 and the sludge suspension was aerated until use.
- Initial biomass concentration: The dry weight was determined from 10 mL of sludge suspension after 0.5 hours of sedimentation. Before the test the sludge was suspended in water up to a concentration of about 4000 mg of sludge dry weight per litre.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
approx. 6 – 8
Nominal and measured concentrations:
- Nominal concentrations: 100, 180, 320, 580 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Aerated apparatus with glass ends
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 500 mL; defined dose of the test or reference substance, 100 mL of deionised 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 deionised water.
- Aeration: Yes, continuous aeration by filtered pressured air (passed through a filter for odour and oil removal)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per abiotic control (replicates): 1; performed at the highest concentration of the test substance and nutrient medium without sludge and with addition of 1 mL of 5 g/L mercury chloride solution.
- Sludge concentration (weight of dry solids per volume): Sludge concentration 18 030 mg/L
- Nutrients provided for bacteria: Synthetic sewage. Synthetic nutrient medium was always prepared fresh on the day of use. The following components were made up to 1 L with deionised water: peptone 16 g, meat extract 11 g, urea ((NH2)2CO) 3 g, sodium chloride (NaCl) 0.7 g, calcium chloride dehydrate (CaCl2.2H2O) 0.4 g, magnesium sulphate heptahydrate (MgSO4.7H2O) 0.2 g and anhydrous dipotassium hydrogen phosphate (K2HPO4) 2.8 g.
- Biomass loading rate: 4000 mg/L: 222 mL/L

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

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Daily
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The measurement of oxygen content was performed in aliquots of the reaction mixtures after the 3 hour incubation period. Bottles with narrow necks and a volume of about 280 mL (BOD bottles) were filled with the sample and positioned on a magnetic stirrer. Then, the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottleneck to avoid contact of the sample with the atmosphere. The decline of oxygen concentration was measured and recorded at 10-minute intervals (respiration rate of activated sludge).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Yes
- Test concentrations: 46, 100, 220, 480 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Details on results:
In the main test, the test substance caused no inhibition of respiration. Abiotic decomposition was not detected even at the highest concentration of the test substance.

VALIDITY CRITERIA
- The respiration rates of two control experiments (at the beginning and at the end of the test) should not differ from each other more than 15 %.
The difference of the respiration rates of control experiments at the beginning and the end of the main test from each other was:
± 0.35 mg/L/h = ± 1.04 % (if average value of two controls 33.35 mg/L/h = 100 %)
± 0.69 mg/L/h = ± 2.10 % (if the value of lower control 33.00 mg/L/h =100 %)

- The EC50 value of the reference substance should be in the range of 5 to 30 mg/L. The EC50 value was 8.1 mg/L (95 % confidence interval: 6.9 to 9.2 mg/L)

The criteria of test validity were fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 8.1 mg/L (95 % confidence interval 6.9 to 9.2 mg/L)
Reported statistics and error estimates:
The values of EC50, EC20 and EC80 for the reference substance were calculated from the equation of regression line prepared by commercial software (Microsoft® Excel 2000).
The confidence intervals of EC50, EC20 and EC80 for the reference substance were calculated by the software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).
The test substance caused no inhibition of respiration rate therefore it was not possible to calculate the EC50 value.

Table 1: Calculation of Inhibition in the Main Test

Substance

Concentration (mg/L)

P1 (mg/L)

P2 (mg/L)

T1 (minutes)

T2 (minutes)

R (mg/L/h)

Inhibition (%)

Control

0

6.46

2.61

3.0

10.0

33.00

0.0

3,5-DCP

20.0

9.08

8.46

1.0

10.0

4.13

87.6

14.0

8.43

7.20

0.5

10.0

7.77

76.7

10.0

8.54

6.56

1.0

10.0

13.20

60.4

7.3

8.09

5.01

0.5

10.0

19.45

41.7

5.2

8.28

4.7

0.5

10.0

22.61

32.2

Test Substance

100

6.42

2.65

3.0

10.0

32.31

3.1

180

6.37

2.77

2.5

9.0

33.23

0.3

320

6.25

2.54

2.0

8.5

34.43

-.3.

580

6.40

2.50

0.5

7.0

36.00

-8.0

1000

6.32

2.67

2.0

8.5

33.69

-1.0

Control

0

627

2.62

3.0

9.5

33.69

0.0

Abiotic Control

1000

8.79

8.74

0.5

10.0

0.32

-

3,5-DCP = 3,5-dichlorophenol

Average R value of the controls = 33.35 mg/L/h (SD = 0.49 mg/L/h)

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the EC50 was determined to be >1000 mg/L.
Executive summary:

The influence of the test substance, Solvent Red 19E, on the respiration rate of activated sludge was investigated after a contact time of 3 hours under GLP conditions.

The 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.

Following a preliminary test using 5 concentrations of the test substance in the range 46 to 1000 mg/L, in the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 were used to investigate the effects of the test substance. Concentrations were 100, 180, 320, 580 and 1000 mg/L. Activated sludge from a municipal sewage treatment plant was exposed to the test material along with two control experiments (one at the beginning and one at the end of the test) an abiotic control and 3,5-dichlorophenol as a reference substance.

In the main test, the test substance caused no inhibition of respiration. Abiotic decomposition was not detected even at the highest concentration of the test substance.

The test met all the validity criteria as the respiration rate of the controls did not differ too greatly and the EC50 of the reference substance was in the appropriate range.

Under the conditions of this study, the EC50 was determined to be >1000 mg/L. All tested concentrations in the main test caused no significant inhibition of respiration rate. Therefore it was not possible to calculate the EC50 value from available data.

Description of key information

The EC50 was determined to be >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The influence of the test substance, Solvent Red 19E, on the respiration rate of activated sludge was investigated after a contact time of 3 hours under GLP conditions.

The 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.

Following a preliminary test using 5 concentrations of the test substance in the range 46 to 1000 mg/L, in the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 were used to investigate the effects of the test substance. Concentrations were 100, 180, 320, 580 and 1000 mg/L. Activated sludge from a municipal sewage treatment plant was exposed to the test material along with two control experiments (one at the beginning and one at the end of the test) an abiotic control and 3,5-dichlorophenol as a reference substance.

In the main test, the test substance caused no inhibition of respiration. Abiotic decomposition was not detected even at the highest concentration of the test substance.

The test met all the validity criteria as the respiration rate of the controls did not differ too greatly and the EC50 of the reference substance was in the appropriate range.

Under the conditions of this study, the EC50 was determined to be >1000 mg/L. All tested concentrations in the main test caused no significant inhibition of respiration rate. Therefore it was not possible to calculate the EC50 value from available data.