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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
The effect of the test substance 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 substance 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
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2016; signature: January 2017
Analytical monitoring:
no
Vehicle:
no
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:
- 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 2.99 g/L, corresponding to 1.50 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 TG 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: For ‘Total Respiration inhibition’ suspended solids 1.50 g per litre (aerobic sludge per litre water); for ‘Inhibition of Heterotrophic Respiration’: suspended solids 1.65 g per litre (aerobic sludge per litre water) and for ‘Measured Inhibition of Nitrification’: suspended solids 1.50 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 Definitive test #1: 20.2 °C ; actual Definitive test #2: 20.9 °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.
In the non-GLP range finder: pH value measured during the study was pH = 6.76 at final (test vessel at 1000 mg/L nominal test item)
In the definitive test #1: ‘Total Respiration inhibition’: pH value of the activated sludge was pH = 7.39 and the pH value of the synthetic waste water was pH = 7.35
In the definitive test #2: ‘Measured Inhibition of Nitrification’: pH value of the activated sludge without ATU was pH = 7.52 and with ATU was pH = 7.57. The pH value of the synthetic waste water was pH = 7.24
Dissolved oxygen:
In the non-GLP range finder: Two replicates were directly sampled after stirring for 10 minutes and two replicates were stirred for 10 minutes and aerated by shaking at 150 rpm for 3 h. Then the flasks were sampled afterwards. Dissolved oxygen concentrations: 100 mg/L nominal test item: 46.43 – 75.85 mg O2 /L before incubation and aeration and 82.19 – 82.17 mg O2/L after 3 hours incubation and aeration.
In the definitive test #1: ‘Total Respiration inhibition’: 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. The flasks was closed with a smokestack with active carbon.
In the definitive test #2: 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. The flasks were closed with polyamide textile and active carbon that allows air exchange to prevent formation of aerosols.
Nominal and measured concentrations:
Preliminary test (non-GLP range finder): 10, 100 and 1000 mg/L nominal (test item); with inhibitions with and without ATU at 2.1, 32 and 1000 mg/L nominal (test item).
For definitive test #1: ‘Total Respiration inhibition’ and : The nominal concentrations tested were: 0 (control), 2.1, 5.6, 32, 180 and 1000 mg/L (in five replicates per concentration). The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (triplicate per concentration)
For definitive test #2: ‘Measured Inhibition of Nitrification’: The nominal concentrations tested were: 0 (control), 1.06, 5.6, 32, 180 and 1000 mg/L (in five replicates per concentration). 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): Open, vessel continuously aerated with seal.
- Material, size, headspace, fill volume: glass, 500 mL fill volume. See table 1 and 3.
- Aeration: Continuously aerated with compressed air.
- 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): Five (negative) and three (reference item)
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)
- Biomass loading rate: See table 1 and 3.

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: pH and temperature were determined in all test media and controls; prior to and at the end of the 3 hour incubation period. and 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
- Test concentrations: See table 1 and 3.
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L nominal (test item); with inhibitions with and without ATU at 2.1, 32 and 1000 mg/L nominal (test item).
- 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:
EC50
Effect conc.:
4.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
306 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 95% C.I.: 245 - 387 mg/L
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
136 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 95% C.I.: 95 - 190 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: No statistically significant inhibition at 32 mg/L (P < 0.001, ANOVA, Dunnett`s Method) for the inhibition of the heterotrophic respiration.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1.39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: 95% C.I.: 1.07 - 4.42 mg/L
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: for copper (II) sulphate: Definitive Test 1 (total respiration inhibition): EC50 102 (95% C.I. 100 - 104) mg/L ; Definitive Test 2 (Inhibition of Nitrification): EC50 97.6 (95% C.I. 95.5 - 99.7) mg/L. N-Methylaniline was additionally tested (with and without ATU) at three different concentrations: 0.1, 1.8 and 32 mg/L and a dilution factor of 18. Full information is provided in the full study report.
- Other: Yes the reference substance results were valid, the EC50 for copper (II) sulphate 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 α-value (acceptable probability of incorrectly concluding that there is a difference) is α =0.05. The EC-values of the test item and the reference item were calculated by biphasic regression and linear regression, respectively, using software GraphPadPrism. Full details of the statistical analysis are provided in the full study report.

Statistically significant inhibition at the lowest tested concentration (P < 0.05, ANOVA, Dunnett`s Method) for the inhibition of total respiration and nitrification was observed. No statistically significant inhibition at 32 mg/L (P < 0.05, ANOVA, Dunnett`s Method) for the inhibition of the heterotrophic respiration was observed.

Table 5. Definitive Test 1 - ‘Total Respiration inhibition’: Oxygen uptake rates and Inhibition of the respiration of the control and test item and reference items

Test
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L h]

Inhibition

[%]

Mean Inhibition
[%]

Control

1

32.4

2

31.2

3

30.4

4

30.4

5

28.4

6

27.6

2.1

1

22.8

24

16

2

27.2

10

3

26.0

14

4

23.2

23

5

28.0

7

5.6

1

14.4

52

47

2

18.8

38

3

15.2

50

4

16.8

44

5

14.8

51

32

1

12.0

60

58

2

13.2

56

3

12.8

57

4

12.4

59

5

12.4

59

180

1

10.8

64

65

2

10.8

64

3

10.8

64

4

9.6

68

5

10.0

67

1000

1

0.8

97

97

2

0.8

97

3

0.0

100

4

0.8

97

5

1.2

96

Repl. = Replicate;

Coefficient of variation of oxygen uptake rates of the control: 5.9 %:
Suspended solids in the test vessel: 1.5 g/L

 

Table 6. Definitive Test 1 : Specific respiration rates of the control replicates

 

 

Repl.

Specific Respiration Rate Rs
[mg O2/g h]

 

Control

1

21.6

2

20.8

3

20.3

4

20.3

5

18.9

6

18.4

Mean value

20.0

Repl. = Replicate

Suspended solids in the test vessel: 1.5 g/L

 

Table 7. Definitive Test 2 - ‘Total Respiration inhibition’ and ‘Inhibition of the Heterotrophic Respiration’ and ‘Inhibition of Nitrification’: Oxygen uptake rates and Inhibition of the respiration of the control and test item and reference items

Total Respiration inhibition

Suspended solids in the test vessel: 1.59 g/L

Test
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L h]

Inhibition

[%]

Mean Inhibition
[%]

Control **1

1

38.5

2

38.1

3

38.1

4

35.5

5

35.3

6

35.2

1.06

1

36.5

1

13

2

27.0

27

3

36.2

2

4

33.5

9

5

27.7

25

5.6

1

17.8

52

52

2

18.5

50

3

17.6

52

4

17.8

52

5

16.7

55

32

1

15.1

59

60

2

15.4

58

3

14.9

60

4

15.4

58

5

13.3

64

180

1

12.9

65

66

2

13.0

65

3

12.0

67

4

12.9

65

5

11.6

68

1000

1

1.9

95

98

2

0.6

98

3

1.6

96

4

0.1

100

5

0.0

100

 

Inhibition of the Heterotrophic Respiration

Note: Suspended solids in the test vessel: 1.65 g/L

Test
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L h]

Inhibition

[%]

Mean Inhibition
[%]

Control + ATU **2

1

17.0

2

16.6

3

17.0

4

16.7

5

15.3

6

17.1

1.06 + ATU

1

17.9

-8

-4

2

17.4

-5

3

17.3

-4

4

17.4

-5

5

16.5

1

5.6 + ATU

1

16.9

-2

-1

2

17.3

-4

3

16.6

0

4

17.0

-2

5

15.7

5

32+ ATU

1

16.3

2

12

2

9.2

45

3

15.0

10

4

18.0

-8

5

14.5

13

180 + ATU

1

14.2

14

18

2

13.9

16

3

13.5

19

4

13.5

19

5

12.9

22

1000 + ATU

1

0.4

98

98

2

0.2

99

3

0.3

98

4

0.8

95

5

0.2

99

 

Inhibition of Nitrification

Note: Suspended solids in the test vessel: 1.65 g/L

Test
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L h]

Inhibition

[%]

Mean Inhibition
[%]

Control

1

21.5

2

21.5

3

21.1

4

18.8

5

20.0

6

18.1

1.06

1

18.6

8

26

2

9.6

52

3

18.9

6

4

16.1

20

5

11.2

45

5.6

1

0.9

96

95

2

1.2

94

3

1.0

95

4

0.8

96

5

1.0

95

32

1

-1.2

106

99

2

6.2

69

3

-0.1

100

4

-2.6

113

5

-1.2

106

180

1

-1.3

106

106

2

-0.9

104

3

-1.5

107

4

-0.6

103

5

-1.3

106

1000

1

1.5

93

98

2

0.4

98

3

1.3

94

4

-0.7

103

5

-0.2

101

Repl. = Replicate

**1: Coefficient of variation of oxygen uptake rates of the control: 4.4 %

**2: Coefficient of variation of oxygen uptake rates of the control: 4.0 %

 

Table 8. Definitive Test 2 - Specific respiration rates of the control replicates

 

 

Repl.

Specific Respiration Rate Rs

Total Respiration
[mg O2/g h]

 

Control

1

24.2

2

24.0

3

24.0

4

22.3

5

22.2

6

22.1

Mean value

23.1

Repl. = Replicate

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3 hour EC50 for total inhibition was 4.88 mg/L. The 3 hour EC50 for heterotrophic respiration inhibition was 306 (C.I. 245 – 387) mg/L. The EC10 was 136 (C.I. 95 – 190) mg/L and the NOEC was 32 mg/L. The 3 hour EC50 nitrification inhibition was 1.39 (C.I. 1.07 – 4.42) mg/L. 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 in accordance with GLP. A definitive test examining total respiration only was carried out under static conditions with the test item concentrations 2.1, 5.6, 32, 180, 1000 mg/L. 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. The results indicated the necessity to determine separately the inhibition of nitrification and heterotrophic respiration. Therefore, a second definitive test was performed including these parameters. The second definitive test including all parameters was used for effect level estimations. Samples of activated sludge (suspended solids 1.59 and 1.65 g/L for total respiration inhibition and inhibition of nitrification, respectively) were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 1.06, 5.6, 32, 180 and 1000 mg/L with and without ATU inhibitor. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. Five replicates were prepared for each concentration and six for the control. 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 mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 23.1 mg O2/g h. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 97.6 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of respiration for the test item replicates ranged from -13 % to 98 %. The mean inhibition of heterotrophic respiration for the test item replicates ranged from -4 % to 98 %. The mean inhibition of nitrification for the test item replicates ranged from - 26 % to 106 %. Under the conditions of the study, the 3 hour EC50 for total inhibition was 4.88 mg/L. The 3 hour EC50 for heterotrophic respiration inhibition was 306 (C.I. 245 – 387) mg/L. The EC10 was 136 (C.I. 95 – 190) mg/L and the NOEC was 32 mg/L. No statistically significant inhibition at 32 mg/L was determined for the inhibition of the heterotrophic respiration. The 3 hour EC50 nitrification inhibition was 1.39 (C.I. 1.07 – 4.42) mg/L. All effect levels were based on nominal test item concentrations.

Description of key information

Total Respiration Inhibition:

3h-EC50 = 4.88 (C.I. -) mg/L (nominal), 20 °C

3h-EC10 = - (C.I. - ) mg/L (nominal), 20°C

NOEC = < 1.06 mg/L (nominal), 20 °C, OECD TG 209, 2017

 

Heterotrophic Respiration Inhibition:

3h-EC50 = 306 (C.I. 245 - 387) mg/L (nominal), 20 °C

3h-EC10 = 136 (C.I. 95 - 190) mg/L (nominal), 20°C

NOEC = 32 mg/L (nominal), 20 °C, OECD TG 209, 2017

 

Nitrification Inhibition:

3h-EC50 = 1.39 (C.I. 1.07 – 4.42) mg/L (nominal)

3h-EC10 = - (C.I. -) mg/L (nominal)

NOEC = < 1.06 mg/L (nominal), 20 °C, OECD TG 209, 2017

 

The nitrification inhibition effect values are suggested to be used for subsequent environmental risk assessment, as the most conservative effect levels.

Key value for chemical safety assessment

EC50 for microorganisms:
1.39 mg/L

Additional information

Key study: OECD TG 209, 2017 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. A definitive test examining total respiration only was carried out under static conditions with the test item concentrations 2.1, 5.6, 32, 180, 1000 mg/L. 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. The results indicated the necessity to determine separately the inhibition of nitrification and heterotrophic respiration. Therefore, a second definitive test was performed including these parameters. The second definitive test including all parameters was used for effect level estimations. Samples of activated sludge (suspended solids 1.59 and 1.65 g/L for total respiration inhibition and inhibition of nitrification, respectively) were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 1.06, 5.6, 32, 180 and 1000 mg/L with and without ATU inhibitor. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. Five replicates were prepared for each concentration and six for the control. 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 mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 23.1 mg O2/g h. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 97.6 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of respiration for the test item replicates ranged from -13 % to 98 %. The mean inhibition of heterotrophic respiration for the test item replicates ranged from -4 % to 98 %. The mean inhibition of nitrification for the test item replicates ranged from - 26 % to 106 %. Under the conditions of the study, the 3 hour EC50 for total inhibition was 4.88 mg/L. The 3 hour EC50 for heterotrophic respiration inhibition was 306 (C.I. 245 – 387) mg/L. The EC10 was 136 (C.I. 95 – 190) mg/L and the NOEC was 32 mg/L. No statistically significant inhibition at 32 mg/L was determined for the inhibition of the heterotrophic respiration. The 3 hour EC50 nitrification inhibition was 1.39 (C.I. 1.07 – 4.42) mg/L. All effect levels were based on nominal test item concentrations.

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