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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
dated May 30, 2008 (updated on 1st March 2016 with Commission Regulation (EC) No 2016/266)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
- Concentrations: 10, 100, 1000 mg/L

- Sampling method: The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time. The treatment sample was not further aerated. The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 5.5 - 10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason more O2 electrode were used. Simultaneous (a maximum of four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in 6 cycles with the available four O2 electrodes. The oxygen consumption rate (in mg O2/L/hour) was determined from the linear part of the respiration curve (in the range between approximately 2 - 7 mg O2/L).

- Sample storage conditions before analysis: no data
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: At the start of the test defined amounts of test item (1 x 3 mg; 1 x 30 mg and 3 x 300 mg, that correspond to 10, 100 and 1000 mg test item/L concentrations) were directly weighed (administered) into each test flask (empty containers that were filled up to a final volume of 300 mL with water and synthetic sewage, just before the inoculation). The subsequent calculations refer to the initial weighed nominal concentration. Concentrations in excess of nominal 1000 mg test item/L were not tested.

- Eluate: Distilled water

- Differential loading: no

- Controls: Two controls containing only tap water, synthetic sewage feed and inoculum were tested in parallel to single test concentration of the test item under identical test conditions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary

- Method of cultivation: 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.

- Preparation of inoculum for exposure: An aliquot of the final sludge suspension was weighed (6.004 g wet weight), dried and the ratio of wet sludge to dry weight (0.4984 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (24 g dry weight that was equivalent to 289 g wet sludge) was suspended in isotonic saline solution (ad. 8 L) to yield a concentration equivalent to about 3 g/L (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage.
The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature (20 ± 2 °C) for about 24 hours (one day) and fed with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.38). pH adjustment of the inoculum was considered not necessary

- Pretreatment: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18.0 - 22.0 °C
pH:
7.29 - 10.25
Dissolved oxygen:
7 - 8.60 mg O2/L
Nominal and measured concentrations:
Nominal concentrations of 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, size, headspace, fill volume: Erlenmeyer bottles of approximately 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): 1
- No. of vessels per vehicle control (replicates): 1
- No. of vessels per abiotic control (replicates): 1
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to test guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED: The respiration rates (total, heterotrophic and nitrification oxygen uptake rates)

TEST CONCENTRATIONS
- Test concentrations: 10, 100 and 1000 mg/L
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:
other: inhibition of oxygen consumption
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of oxygen consumption
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 10.16 mg/L, (95 % confidence limits: 8.60 – 12.11 mg/L).

Table 1: The Oxygen Concentrations and pH Values in the Test Item Treatment Groups and Controls

Identifi-cation

Concentration (mg/L)

Oxygen concentration (mg O2/L)

pH

Start of the 3-hour aeration

End of the 3-hour aeration

Before the addition of inoculum

Start of the 3-hour aeration

End of the 3-hour aeration

CBA

0.00

7.65

7.02

7.40

7.55

8.16

CBB

 

7.64

7.47

7.37

7.59

8.14

CBC

 

7.71

7.64

7.36

7.60

8.14

CBD

 

7.77

7.69

7.37

7.57

8.15

CBE

 

8.29

7.44

7.54

7.46

7.76

CBF

 

8.36

7.27

7.54

7.66

8.12

CBG

 

8.18

7.15

7.54

7.76

8.16

CBH

 

8.39

7.28

7.55

7.80

8.12

CNA

11.6 mg ATU/L

8.40

7.62

7.46

7.65

8.21

CNB

 

8.20

7.09

7.59

7.84

8.13

R1

2 mg 3,5-DCP/L

8.23

7.28

7.31

7.66

7.93

R2

7 mg 3,5-DCP/L

8.08

7.00

7.36

7.59

7.93

R3

24.5 mg 3,5-DCP/L

8.21

7.99

7.29

7.58

7.93

T1

10 mg Test Item/L

8.47

7.42

7.47

8.13

8.12

T2

100 mg Test Item/L

8.13

7.06

8.75

8.51

8.27

T3/A

1000 mg Test Item/L

8.21

8.25

10.24

9.64

9.13

T3/B

 

8.09

8.54

10.27

9.68

9.18

T3/C

 

8.32

8.57

10.35

9.69

9.20

CAA

1000 mg Test Item/L

8.61

8.54

- *

10.28

9.73

CAB

 

8.50

8.23

- *

10.29

9.77

CAC

 

8.43

8.62

- *

10.30

9.68

3,5-DCP: 3,5-dichlorophenol

ATU: N-allylthiourea

Remarks: * At the Abiotic controls no inoculum was added; therefore, one pH measurement was carried out at the start of the test.

Table 2: The Q1, Q2 and the applied Δt values in the Test Item Treatment and Control Groups; the Oxygen Consumption Rate (R), and % Inhibition of R

Identifi-cation

Concentration (mg/L)

Oxygen concentration (mg O2/L)

Δt (min)

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

Average R (mg O2/Lh)

Inhibition of R (%)

Q1

Q2

CBA

0.00

7.02

2.25

5.5

52.04

46.68

0.00

CBB

7.04

2.19

6

48.50

 

 

CBC

7.22

2.03

6.5

47.91

 

 

CBD

7.30

2.27

6.5

46.43

 

 

CBE

7.02

2.35

5.5

50.95

 

 

CBF

7.27

2.04

7.5

41.84

 

 

CBG

7.15

2.27

7

41.83

 

 

CBH

7.28

2.15

7

43.97

 

 

CNA

11.6 mg ATU/L

7.21

2.35

6

48.60

47.80

-2.39

CNB

7.09

2.39

6

47.00

 

 

R1

2 mg 3,5-DCP/L

7.28

2.09

7

44.49

44.49

4.71

R2

7 mg 3,5-DCP/L

7.00

2.14

9.5

30.69

30.69

34.25

R3

24.5 mg 3,5-DCP/L

7.99

6.62

10

8.22

8.22

82.39

T1

10 mg Test Item/L

7.02

2.36

6

46.60

46.60

0.18

T2

100 mg Test Item/L

7.06

2.19

6.5

44.95

44.95

3.70

T3/A

1000 mg Test Item/L

8.25

7.21

10

6.24

6.46

86.16

T3/B

8.54

7.54

10

6.00

 

 

T3/C

8.57

7.38

10

7.14

 

 

CAA

1000 mg Test Item/L

8.54

8.63

10

-0.54

-0.54

101.16

CAB

8.23

8.32

10

-0.54

 

 

CAC

8.62

8.71

10

-0.54

 

 

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.).

3,5-DCP: 3,5-dichlorophenol

ATU: N-allylthiourea

 

Table3: The Specific Respiration Rate (RS) in the Test Item Treatment and Control Groups

Identification

Concentration (mg/L)

Specific Respiration Rate (mg O2/gh)

Average RS (mg O2/gh)

CBA

0.00

34.69

 

CBB

32.33

31.212

CV(%) = 8.34

CBC

31.94

CBD

30.95

CBE

33.96

CBF

27.89

CBG

27.89

CBH

29.31

CNA

11.6 mg ATU/L

32.40

31.87

CV(%) = 2.37

CNB

31.33

R1

2 mg 3,5-DCP/L

29.66

29.66

R2

7 mg 3,5-DCP/L

20.46

20.46

R3

24.5 mg 3,5-DCP/L

5.48

31.07

T1

10 mg Test Item/L

31.07

29.97

T2

100 mg

Test Item/L

29.97

 

T3/A

1000 mg Test Item/L

4.16

 

T3/B

4.00

4.31 * CV(%)= 9.30

T3/C

4.76

CAA

1000 mg Test Item/L

-0.36

-0.36 CV(%) = 0.00

CAB

-0.36

CAC

-0.36

3,5-DCP: 3,5-dichlorophenol

ATU: N-allylthiourea

CV: Coefficient of variation

*: There is a statistically significant difference between the test item treatment (1000 mg/L) and blank control (2-Sample t-Test; 2-sided; α = 0.05)

 

Validity criteria fulfilled:
yes
Conclusions:
Based on measured oxygen consumption values and calculated specific respiration rates, unequivocal test item effect was established at the highest examined concentration of 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate; however the 3-hour EC50 values of the test item is < 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not be determined.
Executive summary:

In a 3-hour pre-test test according to OECD 209 the influence of the test item on the activity of the activated sludge was evaluated 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.

This pre-test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The test was performed without pH adjustment. All validity criteria of the study were met.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect was noticed at 10 and 100 mg/L (the observed slight, 0.18 and 3.70 % inhibitions were considered as being within the biological variability of the applied test system), but the inhibition was in average 86.16 % at 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate. The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test item had unequivocal influence on the pH at the concentrations of 100 and 1000 mg/L. Before inoculum addition the pH of the test item containing test mixtures at these concentration levels was above the acceptable pH 7 - 8 range.

Based on measured oxygen consumption values and calculated specific respiration rates, unequivocal test item effect was established at the highest examined concentration of 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate; however the 3-hour EC50 values of the test item is < 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not be determined.

During the performance of the pre-experiment unequivocal, significant test item effect on the pH was realized especially at the concentrations of 100 and 1000 mg/L.

In conclusion, these pre-test results demonstrate the significant inhibition of oxygen consumption by the test substance in the examined concentration range; therefore, a definite test with parallel test series (with and without pH adjustment) was considered as necessary.

The definite test will be performed under a new study code; the concentration levels to be examined in the definite test will be based on the results of the present pre-experiment.

Description of key information

Based on measured oxygen consumption values and calculated specific respiration rates, unequivocal test item effect was established at the highest examined concentration of 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate; however the 3-hour EC50 values of the test item is < 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not be determined.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

In a 3-hour pre-test test according to OECD 209 the influence of the test item on the activity of the activated sludge was evaluated 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.

This pre-test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The test was performed without pH adjustment. All validity criteria of the study were met.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect was noticed at 10 and 100 mg/L (the observed slight, 0.18 and 3.70 % inhibitions were considered as being within the biological variability of the applied test system), but the inhibition was in average 86.16 % at 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate. The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test item had unequivocal influence on the pH at the concentrations of 100 and 1000 mg/L. Before inoculum addition the pH of the test item containing test mixtures at these concentration levels was above the acceptable pH 7 - 8 range.

Based on measured oxygen consumption values and calculated specific respiration rates, unequivocal test item effect was established at the highest examined concentration of 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate; however the 3-hour EC50 values of the test item is < 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not be determined.

During the performance of the pre-experiment unequivocal, significant test item effect on the pH was realized especially at the concentrations of 100 and 1000 mg/L.

In conclusion, these pre-test results demonstrate the significant inhibition of oxygen consumption by the test substance in the examined concentration range; therefore, a definite test with parallel test series (with and without pH adjustment) was considered as necessary.

The definite test will be performed under a new study code; the concentration levels to be examined in the definite test will be based on the results of the present pre-experiment.