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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 February 2018 to 15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
23 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 - 32 - 100 - 320 - 1000 mg/L were pipetted directly in 1000 mL laboratory bottles. The bottles were closed immediately.
- Differential loading: Yes (direct addition)
- Controls: Yes - test medium without test or reference item
- 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.): No
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Test System: Activated sludge
- Origin: Municipal sewage treatment plant of 31137 Hildesheim, Germany
- Reason for the selection: Non-adapted activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Date of receipt: 14 February 2018
- Dry sludge concentration: 3.06 g/L, corresponding to 1.53 g/L in the test vessel for replicates without ATU and 2.76 g/L, corresponding to 1.38 g/L in the test vessel for replicates with ATU.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
Guideline default
Post exposure observation period:
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.
Test temperature:
18 - 22 °C, actual: 20.1 °C
pH:
7.5 ± 0.5
Dissolved oxygen:
> 60 - 70 % (ASV)
Nominal and measured concentrations:
Nominal Concentrations: 10 - 32 - 100 - 320 - 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL laboratory bottles with screw cap, ISO 4796
- Type (delete if not applicable): closed
- Aeration: Shaking of the laboratory bottles at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable
- No. of vessels per abiotic control (replicates): Not applicable
- Sludge concentration (weight of dry solids per volume): 3.0 g/L ± 10 %.
- Nutrients provided for bacteria: Yes
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (with and without)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic waste water according to OECD Guideline 209
- Particulate matter: Not reported
OTHER TEST CONDITIONS
- Adjustment of pH: Not required
- Photoperiod: Not applicable
- Light intensity: Not reported
- Details on termination of incubation: Not relevant
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate (total respiration) & N-methylaniline (nitrification)
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
22.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % CI (19.7 - 26.2 mg/L)
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
41.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 95 % CI (27.2 - 49.5 mg/L)
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
15.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: 95 % CI (12.5 - 21.7 mg/L)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
76.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % CI (68.1 - 86.7 mg/L)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
145 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 96 % CI (132 - 162 mg/L)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: 95 % CI (32.2 - 62.2 mg/L)
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: Yes
- Adsorption (e.g. of test material to the walls of the test container): Not reported
- Blank controls oxygen uptake rate: 30.4 mg O2/h (cotrol); 16.0 mg O2/h (heterotrophic control); 14.4 mg O2/L (nitrification control)
- Coefficient of variation of oxygen uptake rate in control replicates: 6.7 % (control); 7.6 % (heterotrophic control); 20.2 % (nitrification control)
Results with reference substance (positive control):
- Results with reference substance valid? Yes
The EC50 of the reference item copper (II) sulphate pentahydrate was 98.7 mg/L and is in the range of 53 - 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 -value (acceptable probability of incorrectly concluding that there is a difference) is alpha =0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression (biphasic regression where necessary) and linear regression, respectively, using software GraphPadPrism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

Table 1. Oxygen uptake rates and specific respiration rates of the total respiration of the control

   Replicate  Oxygen concentration [mg O2/L]  Oxygen uptake rate [mg O2/(L x h)]  Specific respiration rate [mg o2/g x h)]
 Control

 1

2

3

4

5

6

 6.94

6.86

6.70

6.23

5.13

6.59

 33.3

31.1

32.4

29.3

28.8

27.5

 21.8

20.3

21.2

19.1

18.8

18.0

     Mean value  30.4  19.9
     Standard deviation  2.1  
     CV (%)  6.7  

Table 2. Oxygen uptake rates and inhibition of the total respiration of the test item concentrations

 Tesrt item concentration 9mg/L)  Replicate  Oxygen Concentration (mg O2/L)  Oxygen uptake rate [mg O2/(l x h)]  Inhibition (%)  Mean inhibition (%)
 10

 1

2

3

4

5

 5.42

5.06

5.60

6.68

6.23

 31.4

31.0

31.7

28.5

30.8

 -3

-2

-4

6

-1

 -1
 32

 1

2

3

4

5

6.09

6.76

6.20

6.82

7.23 

 24.5

24.4

24.0

24.1

25.5

 19

20

21

21

16

19 
 100

 1

2

3

4

5

6.00

6.08

6.09

6.24

6.43 

 13.4

13.5

13.0

10.2

15.4

56

56

57

66

49 

57 
 320

 1

2

3

4

5

 6.87

7.07

6.80

7.19

7.50

 9.3

9.1

8.6

9.1

6.8

 69

70

72

70

78

 72
 1000

 1

2

3

4

5

6.77

6.55

6.65

7.06

6.98 

9.0

8.9

9.0

8.7

9.4 

70

71

70

71

69 

 70

Table 3. Oxygen uptake rates of the heterotrophic respiration of the control replicates with ATU

   Replicate  Oxygen Concentration (mg O2/L)  Oxygen Uptake Rate [mg O2/(l x h)]
 Control

 1

2

3

4

5

6

5.93

7.62

6.04

7.98

7.95

8.11 

15.4

13.8

16.0

17.6

16.7

16.7

     Mean value  16.0
     Standard deviation  1.2
     CV (%)  7.6

Table 4. Oxygen uptake rates and inhibition of the heterotrophic respiration of the test item concentrations

 Test item concentration (mg/L)  Replicate  Oxygen concentration (mg O2/L)  Oxygen uptake rate (mg O2/L.h)  Inhibition (%)  Mean inhibition (%)
 10 + ATU

 1

2

3

4

5

 5.92

6.10

5.92

5.98

5.79

 14.5

14.8

14.6

15.2

13.4

 9

8

9

5

16

 9
 32 + ATU

1

2

3

4

5

 6.87

6.86

6.47

6.90

6.79

 14.9

15.4

15.7

15.1

15.5

 7

4

2

6

3

 4

 100 + ATU

1

2

3

4

5

 7.29

7.13

7.01

7.29

7.58

10.2

10.1

10.4

10.1

10.6 

 36

37

35

37

34

 36

 320 + ATU

1

2

3

4

5

7.66

7.82

7.81

7.77

7.95 

 6.6

5.9

6.5

6.4

4.8

 59

63

59

60

70

62 

 1000 + ATU

1

2

3

4

5

7.10

7.17

6.73

6.87

7.02 

9.3

9.3

10.2

13.3

14.9 

 42

42

36

17

7

29 

Table 5. Calculated oxygen uptake rates of the nitrification of the control

   Replicate  Oxygen uptake rate (mg o2/(Lxh))
 Control

 1

2

3

4

5

6

 17.9

17.3

16.4

11.6

12.1

10.8

   Mean value  14.4
   Standard deviation  2.9
   CV [%]  20.2

Table 6. Oxygen uptake rates and inhibition of the nitrification of the test item concentrations

Test item concentration (mg/L)   Replicate  Oxygen uptake rate (mg O2/L.h)  Inhibition (%)  Mean Inhibition (%)
 10

 1

2

3

4

5

16.9

16.2

17.1

13.3

17.4 

 -17

-13

-19

8

-21

 -12
 32

 1

2

3

4

5

 9.6

9.0

8.3

9.0

10.0

 33

38

42

38

31

 36
 100

 1

2

3

4

5

3.2

3.4

2.6

0.2

4.8 

 78

76

82

99

67

 80
 320

 1

2

3

4

5

 2.7

3.2

2.1

2.7

2.0

 81

78

85

81

86

 82
 1000

 1

2

3

4

5

 -0.3

-0.4

-1.2

-4.6

-5.5

 102

103

108

132

138

117 
Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item for total respiration and nitrification is 10 mg/L.
The EC10 for total respiration is 22.8 mg/L and the EC50 is 76.5 mg/L.
The NOEC for heterotrophic respiration is 32 mg/L.
The EC10 for heterotrophic respiration is 41.4 mg/L and the EC50 is 145 mg/L.
The EC10 for nitrification is 15.9 mg/L and the EC50 is 46.0 mg/L.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 mg/L with and without ATU. 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 mean inhibition of total respiration for the test item replicates ranged from - 1 % to 72 % (Table 9). The mean inhibition of the heterotrophic respiration for the test item replicates ranged from 4 % to 62 % (Table 11). The mean inhibition of nitrification for the test item replicates ranged from - 12 % to 117 % (Table 13).

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate and N-Methylaniline (with and without ATU) as reference item and the reference toxicity was determined. The EC50-value for the reference item copper (II) sulphate pentahydrate was 98.7 mg/L. The EC50-value for the reference item N-Methylaniline was

1.50 mg/L.

The NOEC of the test item for total respiration and nitrification is 10 mg/L.

The EC10 for total respiration is 22.8 mg/L and the EC50 is 76.5 mg/L.

The NOEC for heterotrophic respiration is 32 mg/L.

The EC10 for heterotrophic respiration is 41.4 mg/L and the EC50 is 145 mg/L.

The EC10 for nitrification is 15.9 mg/L and the EC50 is 46.0 mg/L.

Description of key information

OECD 209; Anon. (2018):

The NOEC for total respiration and nitrification is 10 mg/L.

The EC10 for total respiration is 22.8 mg/L and the EC50 is 76.5 mg/L.

The NOEC for heterotrophic respiration is 32 mg/L.

The EC10 for heterotrophic respiration is 41.4 mg/L and the EC50 is 145 mg/L.

The EC10 for nitrification is 15.9 mg/L and the EC50 is 46.0 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
46 mg/L
EC10 or NOEC for microorganisms:
15.9 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 mg/L with and without ATU. 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 mean inhibition of total respiration for the test item replicates ranged from - 1 % to 72 % (Table 9). The mean inhibition of the heterotrophic respiration for the test item replicates ranged from 4 % to 62 % (Table 11). The mean inhibition of nitrification for the test item replicates ranged from - 12 % to 117 % (Table 13).

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate and N-Methylaniline (with and without ATU) as reference item and the reference toxicity was determined. The EC50-value for the reference item copper (II) sulphate pentahydrate was 98.7 mg/L. The EC50-value for the reference item N-Methylaniline was

1.50 mg/L.

The NOEC of the test item for total respiration and nitrification is 10 mg/L.

The EC10 for total respiration is 22.8 mg/L and the EC50 is 76.5 mg/L.

The NOEC for heterotrophic respiration is 32 mg/L.

The EC10 for heterotrophic respiration is 41.4 mg/L and the EC50 is 145 mg/L.

The EC10 for nitrification is 15.9 mg/L and the EC50 is 46.0 mg/L.

As nitrifying bactrial populations were the most sensitive to the test item in this test, the nitrification effect values were used for chemical safety assessment.

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