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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-01-10 to 2017-02-08
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)
Version / remarks:
2016-03-01
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010-07-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
In this study no analytical measurements were performed. Because of the directly added test item the obtained results were referred to the nominal test item concentrations.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Just before the start of the test defined amounts (5 x 300; 5 x 120; 5 x 48; 5 x 19.2; 5 x 7.7 and 5 x 3.1 mg) of the test item were administered directly into empty containers that were filled up with water and synthetic sewage, just before the inoculation to obtain a final test item concentrations of 1000, 400, 160, 64, 25.6 and 10.24 mg/L per replicate. Concentrations in excess of nominal 1000 mg test item/L were not tested.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 48 hours (2 days) and was fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.63) and before use (pH: 7.75).
- Name and location of sewage treatment plant where inoculum was collected: Sewage plant for domestic sewage in Balatonfüred, Hungary
- Preparation of inoculum for exposure: An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis). At the concentration calculation the dilution resulted by the fed synthetic sewage was taken into consideration.
- Pretreatment: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
- Initial biomass concentration: 3 g per litre (on dry weight basis)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
no data
Test temperature:
20.5 - 21.7 °C
pH:
7.89 - 8.75
Dissolved oxygen:
7.07 - 8.25 mg O2/L
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 10.24, 25.6, 64, 160, 400 and 1000 mg/L.
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, size, headspace, fill volume: glass, 300 mL volume
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 4 for the start control group, 4 for the end control group
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g per litre (on dry weight basis).
- Nitrification inhibitor used: N-allylthiourea, 11.6 mg/L
- Nutrients provided for bacteria: 9.6 mL synthetic sewage mixture per vessel was provided before the inoculation

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

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: 3 concentrations tested: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: In the preliminary test the nitrification respiration was insignificant and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification is occurred. Therefore the main test was performed in one set of test vessels, without ATU addition. In the main test (for informative reason) a nitrification control group (containing N-allylthiourea) was investigated with three parallels. The abiotic control group in the preliminary experiment did not show oxygen uptake therefore in the main test abiotic control group was not included.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
732.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
55.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
25.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption: no
- Blank controls oxygen uptake rate: average - 50.2 mg O2/Lh
- Coefficient of variation of oxygen uptake rate in control replicates: not determined
Results with reference substance (positive control):
- Results with reference substance valid? Yes
Nominal test concentrations: 2, 7 and 24.5 mg/L
- Relevant effect levels: EC50 = 16.3 mg/L (95 % confidence limits: 13.1-20.3 mg/L)

Table 1.:The Oxygen Consumption Rate (R), Q1, Q2 and the applied Δt valuesin the Blank Control and Test Item Treatment Groups

Test group
No.

Identifi-cation

Concentration
(mg/L)

Oxygen concentration
(mg O2/L)

Δt (min)

Oxygen Consumption Rate (R)
(mg O2/Lh)

Average R

(mg O2/Lh)

Q1

Q2

1, 12

CBA

0.00

7.25

2.04

6.5

48.09

50.20

CBB

7.20

2.23

6

49.70

CBC

7.28

2.02

6.5

48.55

CBD

7.09

2.38

5.5

51.38

CBE

7.39

2.05

6.5

49.29

CBF

7.30

2.26

6

50.40

CBG

7.37

2.08

6

52.90

CBH

7.07

2.37

5.5

51.27

6

T1A

10.24

7.00

2.36

5.5

50.62

48.48

T1B

7.01

2.37

6

46.40

T1C

7.24

2.33

6

49.10

T1D

7.21

2.11

6.5

47.08

T1E

7.39

2.06

6.5

49.20

7

T2A

25.6

7.15

2.27

6.5

45.05

47.37

T2B

7.35

2.10

6.5

48.46

T2C

7.16

2.36

6

48.00

T2D

7.29

2.29

6.5

46.15

T2E

7.24

2.32

6

49.20

8

T3A

64

7.31

2.37

6.5

45.60

43.82

T3B

7.29

2.27

7

43.03

T3C

7.33

2.01

7.5

42.56

T3D

7.22

2.20

7

43.03

T3E

7.21

2.35

6.5

44.86

9

T4A

160

7.01

2.32

7

40.20

40.07

T4B

7.25

2.31

7.5

39.52

T4C

7.10

2.30

7.5

38.40

T4D

7.18

2.02

7.5

41.28

T4E

7.07

2.29

7

40.97

10

T5A

400

7.09

2.23

8.5

34.31

35.01

T5B

7.08

2.10

8.5

35.15

T5C

7.04

2.26

8

35.85

T5D

7.10

2.26

8.5

34.16

T5E

7.07

2.03

8.5

35.58

11

T6A

1000

7.80

4.94

10

17.16

17.90

T6B

7.02

4.20

10

16.92

T6C

8.25

4.63

10

21.72

T6D

7.02

3.62

10

20.40

T6E

7.80

5.58

10

13.32

Q1:   the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);

Q2:   the oxygen concentration at the end of the selected section of the linear phase (mg/L);

Δt:   the time interval between these two measurements (min.).

 

Table 2.:The Specific Respiration Rate (RS)in the Blank Control and Test ItemTreatment Groups

Test group
No.

Identification

Concentration
(mg/L)

Specific Respiration Rate
(mg O2/gh)

Average RS
(mg O2/gh)

1, 12

CBA

0.00

32.06

33.47

CV(%)= 3.20

CBB

33.13

CBC

32.37

CBD

34.25

CBE

32.86

CBF

33.60

CBG

35.27

CBH

34.18

6

T1A

10.24

33.75

32.32n.s.

 

CV(%)= 3.54

T1B

30.93

T1C

32.73

T1D

31.38

T1E

32.80

7

T2A

25.6

30.03

31.58n.s.

 

CV(%)= 3.63

T2B

32.31

T2C

32.00

T2D

30.77

T2E

32.80

8

T3A

64

30.40

29.21 *

 

CV(%)= 3.04

T3B

28.69

T3C

28.37

T3D

28.69

T3E

29.91

9

T4A

160

26.80

26.72 *

 

CV(%)= 2.90

T4B

26.35

T4C

25.60

T4D

27.52

T4E

27.31

10

T5A

400

22.87

23.34 *

 

CV(%)= 2.15

T5B

23.44

T5C

23.90

T5D

22.78

T5E

23.72

11

T6A

1000

11.44

11.94 *

 

CV(%)= 18.38

T6B

11.28

T6C

14.48

T6D

13.60

T6E

8.88

* :  Statistically significantly different compared to the control (Bonferroni t-Test; α = 0.05)

n.s.:  Statistically not significant significantly different compared to the control (Bonferroni t-Test; α = 0.05)

 

Table 3.:The Percentage Inhibition (IT) of Total Oxygen Consumption

Test group
No.

Identification

Test group

Concentration
(mg/L)

Inhibition (IT), of total oxygen consumption
(%)

1, 12

CB (A-H)

Blank Control

0.00

2

CN (A-C)

Nitrification Control

11.6

1.39

3

R1(A-C)

Reference Item

2

5.82

4

R2(A-C)

Reference Item

7

19.37

5

R3(A-C)

Reference Item

24.5

66.02

6

T1(A-E)

Test Item

10.24

3.43

7

T2(A-E)

Test Item

25.6

5.63

8

T3(A-E)

Test Item

64

12.72

9

T4(A-E)

Test Item

160

20.17

10

T5(A-E)

Test Item

400

30.26

11

T6(A-E)

Test Item

1000

64.33

 

Validity criteria fulfilled:
yes
Conclusions:
In an activated sludge respiration inhibition test the EC50 value of SIKA Hardener AI was determined to be 732.6 mg/L (95% confidence limits: 517.8–1036.6 mg/L) and the EC10 was determined to be 55.3 mg/L (CI: 38.8 - 79.0 mg/L) . Based on the statistical and biological evaluation in this test the NOEC was determined as 25.6 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item Sika Hardener AI on the activity of the activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item Sika Hardener AI was investigated at the nominal concentrations of 10.24; 25.6; 64; 160; 400 and 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test was performed without pH adjustment. All validity criteria of the study were met. The inhibition of the oxygen consumption rates showed clear dose-related tendency in the whole examined concentration range 10.24-1000 mg/L. At the concentration of 10.24 mg/L 3.4 %, at 25.6 mg/L 5.6 % inhibitions were calculated. At the concentration of 64 mg/L 12.7 % , at 160 mg/L 20.2 %, at 400 mg/L 30.3 %, at the highest examined concentration of 1000 mg/L 64.3 % inhibitions were obtained. The degree of inhibition (64.3 %) at the highest examined concentration level of 1000 mg/L allowed the calculation of the 3-hour exact EC10 and EC50 values. Based on the available data the 3-hour EC10, EC50 and their 95 % confidence limits were calculated. The EC50 value was determined as 732.6 mg/L (CI: 517.8 - 1036.6), consequently EC50>100 mg/L, and EC10 as 55.3 mg/L (CI: 38.8–79.0).

Description of key information

In an activated sludge respiration inhibition test the EC50 value of SIKA Hardener AI was determined to be 732.6 mg/L (95% confidence limits: 517.8–1036.6 mg/L) and the EC10 was determined to be 55.3 mg/L (CI: 38.8 - 79.0 mg/L) . Based on the statistical and biological evaluation in this test the NOEC was determined as 25.6 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
732.6 mg/L
EC10 or NOEC for microorganisms:
55.3 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item Sika Hardener AI on the activity of the activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item Sika Hardener AI was investigated at the nominal concentrations of 10.24; 25.6; 64; 160; 400 and 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test was performed without pH adjustment. All validity criteria of the study were met. The inhibition of the oxygen consumption rates showed clear dose-related tendency in the whole examined concentration range 10.24-1000 mg/L. At the concentration of 10.24 mg/L 3.4 %, at 25.6 mg/L 5.6 % inhibitions were calculated. At the concentration of 64 mg/L 12.7 % , at 160 mg/L 20.2 %, at 400 mg/L 30.3 %, at the highest examined concentration of 1000 mg/L 64.3 % inhibitions were obtained. The degree of inhibition (64.3 %) at the highest examined concentration level of 1000 mg/L allowed the calculation of the 3-hour exact EC10 and EC50 values. Based on the available data the 3-hour EC10, EC50 and their 95 % confidence limits were calculated. The EC50 value was determined as 732.6 mg/L (CI: 517.8 - 1036.6), consequently EC50>100 mg/L, and EC10 as 55.3 mg/L (CI: 38.8–79.0).