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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-03-2018 to 14-01-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
The effect of the test item on the inhibition of respiration rate of aerobic wastewater microorganisms of activated sludge was investigated. The test organisms were exposed to a series of test concentrations of the test item for 3 hours and the effects on oxygen up take were measured. Results are reported relative to the reference standard controls: copper (II) sulphate pentahydrate.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2016; signature: January 2017
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct pipetting of the test item into 1000 mL test vessels. With shaking at 150 rpm
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: (Blank) Controls: were untreated ; Concurrent reference item: Copper (II) Sulphate controls were added by addition to test vessels from a 300 g/L stock solution to test concentrations: 58, 100 and 180 mg/L in triplicate.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: aerobic activated sludge from 31137 Hildesheim, Germany municipal sewage treatment plant
- Method of cultivation: See below.
- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %.
- Pretreatment: The sludge was used within 24 h after sampling. The dry sludge concentration was 3.16 g/L, corresponding to 1.58 g/L in the test vessel. The test item was not pretreated. The reference item was ultrasonicated.
- Other: Synthetic waste water was prepared according to OECD 209. The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
- Initial biomass concentration: suspended solids 1.58 g per litre (aerobic sludge per litre water).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
nominal 18 - 22 or 20 ± 2°C; actual: 19.8 °C
pH:
The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
pH value measured during the study was pH = 6.70 at final (test vessel 1 at 1000 mg/L nominal test item) in the preliminary non-GLP test. Therefore, not measured during the definitive test.
Dissolved oxygen:
Dissolved oxygen concentrations: 100 mg/L nominal test item: 2.69 – 3.13 mg O2 /L after incubation and aeration in the preliminary non-GLP test.
Aeration was employed by shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
Nominal and measured concentrations:
Preliminary test:
10, 100 and 1000 mg/L nominal (test item)
Additional replicate of 10, 100 and 1000 mg/L nominal (test item) and addition of ATU (inhibition of nitrification)
Definitive test:
The nominal concentrations tested were: 0 (control), 1.0, 5.6, 32.0, 180.0 and 1000.0 mg/L (in five replicates per concentration; six for control)
The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (triplicate per concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flasks
- Type (delete if not applicable): Closed, screw cap aerated by shaking at 150 rpm
- Material, size, headspace, fill volume: See table 1.
- Aeration: By shaking at 150 rpm only.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Six (negative) and one (reference item)
- Biomass loading rate: See table 1.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage water was in accordance with OECD TG 209
- Culture medium different from test medium: Yes, chlorine free tap water used for culture medium; synthetic wastewater and RO used for test medium. Replaced during inoculum pretreatment stage and media preparation.
- Intervals of water quality measurement: Temperatures were determined in all test media and controls; prior to and at the end of the 3 hour incubation period. pH was determined prior to testing. Dissolved oxygen values was determined in the 100 mg/L test item vessels only and/or within preliminary test.

OTHER TEST CONDITIONS
- Adjustment of pH: No.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase. The oximeter instrument measured the oxygen depletion for at least 3 minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5.6
- Justification for using fewer concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations:
Preliminary test: 10, 100 and 1000 mg/L nominal (test item) and additional replicate of 10, 100 and 1000 mg/L nominal (test item) and addition of ATU (inhibition of nitrification)
Definitive test:
The nominal concentrations tested were: 0 (control), 1.0, 5.6, 32.0, 180.0 and 1000.0 mg/L (in five replicates per concentration; six for control)
The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (triplicate per concentration)
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
copper (II) sulphate pentahydrate
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
Remarks:
respiration rate
Remarks on result:
other: No statistically significant inhibition, ANOVA, Dunnett's Method
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
29.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% C.I.: 16.1 - 94.6
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
38.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% C.I.: 32.1 - 103
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
69.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% C.I.: 39.5 - 152
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% C.I.: -
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable.
- Adsorption (e.g. of test material to the walls of the test container): None reported.
- Blank controls oxygen uptake rate: Range: 19.1 to 30.5 mg O2 / L per hour and mean: 18.8 mg O2 / L per hour. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50 value of the reference item was in the required range. Therefore, no impact on the results of the test item was expected through lower respiration.
- Coefficient of variation of oxygen uptake rate in control replicates: 1.58% (Required: CV < 30%).
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: EC50 76.2 (95% C.I. 74.4 -77.8) mg/L
- Other: Yes the reference substance results were valid, the EC50 for copper (II) sulphate was between 74.4 and 77.8 mg/L (total respiration) and was within the expected range: 53 to 155 mg/L.
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One-Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-value of the test item and reference item were calculated by sigmoidal response and linear regression using software: GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures, when possible. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated using MS Excel.

Table 3. Oxygen uptake rates and Inhibition of the respiration of the control and test item and reference items

Test Item Concentration*

Repl.

Oxygen
Concentration**

Oxygen Uptake Rate R

Inhibition

Mean Inhibition

[mg/L]

 

[mg O2/L]

[mg O2/(L×h)]

[%]

 [%]

Control

1

5.71

30.5

19.3

-

2

5.87

30.1

19.1

3

5.34

29.5

18.7

4

6.22

29.2

18.5

5

6.03

29.6

18.7

6

6.37

29.2

18.5

1

1

6.67

31.0

-4

-3

2

6.10

30.8

-4

3

6.14

31.6

-6

4

7.01

26.9

9

5

6.86

33.3

-12

5.6

1

5.64

25.3

15

12

2

5.20

27.9

6

3

5.04

26.9

9

4

5.31

23.8

20

5

5.68

27.0

9

32

1

6.56

27.1

9

12

2

6.50

26.3

11

3

5.60

22.9

23

4

7.18

26.9

9

5

6.23

26.9

9

180

1

6.16

11.6

61

64

2

6.31

11.7

61

3

6.23

10.8

64

4

6.52

7.3

75

5

6.52

11.8

60

1000

1

8.07

11.2

62

64

2

6.89

11.4

62

3

6.43

11.3

62

4

6.84

8.9

70

5

6.99

11.4

62

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 1.58%

Suspended solids in the test vessel: 1.58 g/L.

 

Table 4. Specific respiration rates of the control replicates

 

Repl.

Oxygen
Concentration*

Oxygen Uptake Rate R

Specific Respiration Rate Rs

 

 

[mg O2/L]

[mg O2/(L×h)]

[mg O2/g×h]

Control

1

5.71

30.5

19.3

2

5.87

30.1

19.1

3

5.34

29.5

18.7

4

6.22

29.2

18.5

5

6.03

29.6

18.7

6

6.37

29.2

18.5

 

Mean value

29.7

18.8

Standard deviation

0.47

 

CV [%]

1.58

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 1.58%

Suspended solids in the test vessel: 1.58 g/L.

 

The mean specific respiration rate (Rs) of the control cultures was ca. 20 mg O2/gh (actual 18.8mg O2/g h). The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50 value of the reference item was in the required range. Therefore, no impact on the results of the test item was expected through lower respiration. The oxygen consumption rates of the two negative (blank; inoculum) controls differed by 1.58 % therefore meeting guideline the validity criteria (< 30%). The reference substance results were valid, the EC50 for copper (II) sulphate was (total respiration): actual 76.2 mg/L and was within the expected range: 53 to 155 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3-hour EC50 for total inhibition was 69.7 (C.I. 39.5 – 152) mg/L. The EC20 was 38.6 (C.I. 32.1 – 103) mg/L, the EC10 was 29.8 (C.I. 16.1 – 94.6) mg/L. The NOEC was 32 mg/L with no statistically significant inhibition of respiration rates below this concentration. All effect levels were based on nominal test item concentrations.
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 under GLP. Following a preliminary range finding test at 10, 100 and 1000 mg/L concentration which indicated no inhibition of nitrification, a definitive test was conducted examining total respiration only was carried out under static conditions. Samples of activated sludge suspended solids 1.58 g/L, were exposed to dilutions of the test item for three hours under static conditions with the concentrations: 1, 5.6, 32, 180 and 1000 mg/L. Five replicates were prepared for each concentration and six for the control. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 76.2 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 18.8 mg O2/gh and Coefficient of Variation of 1.58%. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50 value of the reference item was in the required range. The mean inhibition of respiration for the test item replicates ranged from -3% to 64%. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the concentration of 32 mg/L. The EC10 was 29.8 (C.I. 16.1 – 94.6) mg/L and the EC20 was 38.6 (C.I. 32.1 – 103) mg/L. The EC50 was 69.7 (C.I. 39.5 – 152) mg/L.

Description of key information

EC50 = 69.7 (C.I. 39.5 – 152) mg/L (nominal), 20 °C, OECD TG 209, 2019

EC20 = 38.6 (C.I. 32.1 – 103) mg/L (nominal), 20 °C, OECD TG 209, 2019

EC10 = 29.8 (C.I. 16.1 – 94.6) mg/L (nominal), 20°C, OECD TG 209, 2019

NOEC = 32 mg/L (nominal), 20 °C, OECD TG 209, 2019

Key value for chemical safety assessment

EC50 for microorganisms:
69.7 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information

Key study : OECD TG 209, 2019 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 under GLP. Following a preliminary range finding test at 10, 100 and 1000 mg/L concentration which indicated no inhibition of nitrification, a definitive test was conducted examining total respiration only was carried out under static conditions. Samples of activated sludge suspended solids 1.58 g/L, were exposed to dilutions of the test item for three hours under static conditions with the concentrations: 1, 5.6, 32, 180 and 1000 mg/L. Five replicates were prepared for each concentration and six for the control. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 76.2 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 18.8 mg O2/gh and Coefficient of Variation of 1.58%. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50 value of the reference item was in the required range. The mean inhibition of respiration for the test item replicates ranged from -3% to 64%. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the concentration of 32 mg/L. The EC10 was 29.8 (C.I. 16.1 – 94.6) mg/L and the EC20 was 38.6 (C.I. 32.1 – 103) mg/L. The EC50 was 69.7 (C.I. 39.5 – 152) mg/L.