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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.09.2019 - 26.11.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
May 30, 2008 (updated on 1st March 2016)
Deviations:
no
Qualifier:
according to
Guideline:
other: OCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test
Version / remarks:
January 2012
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:
Just before the start of the test defined amounts of the test item corresponding to the concentrations were administered directly into empty containers that were filled up with water and synthetic sewage (to a final volume of 300 mL), just before the inoculation.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Balatonfüred, Hungary on 30 September 2019
- Preparation of Activated Sludge Inoculum: 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. An aliquot of the final sludge suspension was weighed (5.072 g wet weight), dried and the ratio of wet sludge to dry weight (0.4688 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (36 g dry weight that was equivalent to 85.60 g wet sludge) was suspended in isotonic saline solution (ad. 12 L) to yield a concentration equivalent to about 3 g per litre (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 activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.65), additional pH adjustment of the inoculum was considered as not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.0 - 20.7 °C
pH:
7.62 - 9.50 (discussion see below)
Dissolved oxygen:
7.03 - 8.04 mg/L
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320, 640, 1000 mg/L
No measured concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, fill volume: glass, 300 mL
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per abiotic control (replicates): 3
- Nitrification inhibitor used: N-allylthiourea

OTHER TEST CONDITIONS
- Adjustment of pH: yes. In the present experiment two test series were investigated: one series without pH adjustment (10-1000 mg/L), one series with additional pH adjustment (320-1000 mg/L).

EFFECT PARAMETERS MEASURED
The oxygen concentration was measured with O2 electrode (working based on LDO (Luminescent Dissolved Oxygen) method) after the incubation.
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations, reference item concentrations and controls. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes, concentrations and pH adjustment
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:
inhibition of total respiration
Remarks on result:
other: with pH adjustment
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: with pH adjustment
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: with pH adjustment
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
772.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without pH adjustment
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without pH adjustment
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
362.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without pH adjustment
Details on results:
The pH of the test mixtures (before inoculum addition) remained within the required range of pH 7 – 8 at the concentrations of 10 and 32 mg/L. Without pH adjustment the pH of test mixtures (before inoculum addition) varied between 8.52 - 8.55 at 100 mg/L, between 9.71 - 9.73 at 320 mg/L, between 10.29-10.31 at 640 mg/L and it varied between 10.54 - 10.58 at 1000 mg/L. Inhibition was not noticed in the concentrations of 10 and 32 mg/L concentrations. The obtained 8.75 % inhibition at the concentration of 100 mg/L was evaluated rather as being within the biological variability range of the applied test system, than a test item effect. No inhibition was noticed at 320 mg/L at both conditions: with and without pH setting. Without pH setting 36.34 % inhibition was obtained at 640 mg/L and 76.71 % at 1000 mg/L. No inhibition was noticed in the whole examined concentration range up to 1000 mg/L, with pH setting.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) should be in the range of 2 mg/L to 25 mg/L for total respiration, 5 mg/L to 40 mg/L for heterotrophic respiration and 0.1 mg/L to 10 mg/L for nitrification respiration.

Table 1: The Q1, Q2 and the applied Δt values,theOxygen Consumption Rate (R), and % Inhibition of Rin the Test Item Treatment Groups

Identification

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

T1A

10 mg
Test Item/L

7.07

2.06

6

50.10

47.56

-1.60

T1B

7.16

2.34

6

48.20

T1C

7.24

2.12

7.5

40.96

T1D

7.34

2.34

6

50.00

T1E

7.32

2.06

6.5

48.55

T2A

32 mg
Test Item/L

7.13

2.22

6.5

45.32

46.66

0.33

T2B

7.03

2.20

6.5

44.58

T2C

7.17

2.23

6.5

45.60

T2D

7.12

2.23

6

48.90

T2E

7.28

2.39

6

48.90

T3A

100 mg
Test Item/L

7.29

2.04

7.5

42.00

42.72

8.75

T3B

7.20

2.25

6.5

45.69

T3C

7.25

2.03

8

39.15

T3D

7.01

2.03

8

37.35

T3E

7.07

2.13

6

49.40

T4A

320 mg
Test Item/L

7.40

2.20

6

52.00

47.05

-0.50#

T4B

7.10

2.14

7

42.51

T4C

7.27

2.36

7

42.09

T4D

7.19

2.01

6.5

47.82

T4E

7.05

2.39

5.5

50.84

T5A

640 mg
Test Item/L

7.49

2.46

10

30.18

29.81

36.34

T5B

7.41

2.83

9.5

28.93

T5C

7.33

2.84

10

26.94

T5D

7.01

2.20

9.5

30.38

T5E

7.13

2.24

9

32.60

T6A

1000 mg
Test Item/L

7.73

6.06

9.5

10.55

10.90

76.71

T6B

7.38

5.83

10

9.30

T6C

7.64

5.79

9.5

11.68

T6D

7.90

5.90

10

12.00

T6E

7.78

6.04

9.5

10.99

T4pHA

320 mg
Test Item/L

7.20

2.17

6.5

46.43

46.77

0.09

T4pHB

7.33

2.41

6

49.20

T4pHC

7.19

2.03

6.5

47.63

T4pHD

7.27

2.03

7

44.91

T4pHE

7.18

2.23

6.5

45.69

T5pHA

640 mg
Test Item/L

7.03

2.13

6.5

45.23

47.55

-1.57#

T5pHB

7.06

2.14

6.5

45.42

T5pHC

7.11

2.29

6

48.20

T5pHD

7.08

2.01

6.5

46.80

T5pHE

7.42

2.21

6

52.10

T6pHA

1000 mg
Test Item/L

7.05

2.16

6

48.90

47.39

-1.22#

T6pHB

7.23

2.21

6.5

46.34

T6pHC

7.31

2.23

6.5

46.89

T6pHD

7.27

2.14

6.5

47.35

T6pHE

7.29

2.15

6.5

47.45

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.).

#:       the negative values were considered as zero inhibition.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item 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 that the test item caused effect on the activated sludge inoculum, the test item was investigated at the nominal concentrations of 10, 32, 100, 320, 640 and 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. These test item concentrations were tested without pH adjustment. Based on the preliminary experience, the test item affects adversely the pH within the test system; therefore, a second series of test containers were investigated at the concentrations of 320, 640 and 1000 mg/L. In these additional containers pH adjustment (an additional neutralization step to the required pH range of 7-8) was performed with 1N HCl prior to inoculum addition. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L ran; 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. All validity criteria of the study were met.The pH of the test mixtures (before inoculum addition) remained within the required range of pH 7 – 8 at the concentrations of 10 and 32 mg/L. Without pH adjustment the pH of test mixtures (before inoculum addition) varied between 8.52 - 8.53 at 100 mg/L, between 9.71 - 9.73 at 320 mg/L, between 10.29-10.31 at 640 mg/L and it varied between 10.54 - 10.58 at 1000 mg/L. Inhibition was not noticed in the concentrations of 10 and 32 mg/L concentrations. The obtained 8.75 % inhibition at the concentration of 100 mg/L was evaluated rather as being within the biological variability range of the applied test system, than a test item effect. No inhibition was noticed at 320 mg/L at both conditions: with and without pH setting. Without pH setting 36.34 % inhibition was obtained at 640 mg/L and 76.71 % at 1000 mg/L. No inhibition was noticed in the whole examined concentration range up to 1000 mg/L, with pH setting. The degree of inhibition of oxygen consumption rates (36.34 and 76.71 %) obtained without pH adjustment at the concentration levels of 640 and 1000 mg/L allowed the calculation of the 3-hour exact EC10, EC50 values and estimation of EC80 values. Based on the available data the 3-hour EC10, EC50 and EC80 values and their 95 % confidence limits were calculated (in the case of EC80 estimated) by Probit analysis. The values are the following:

EC10 : 362.5 mg/L    (95 % conf. limits: 33.0–538.8 mg/L),

EC50 : 772.4 mg/L    (95 % conf. limits: 597.8–1073.0 mg/L),

EC80 : 1041.6 mg/L  (95 % conf. limits: 828.8–1563.7 mg/L).

Without pH adjustment, the EC50 value was determined as 772.4mg/L, consequently1000 mg/L > EC50> 100 mg/L.

With additional neutralization step no inhibition was noticed up to 1000 mg/L. In this case the available data did not allow the calculation of exact ECx values.

With additional neutralization step (before the inoculation) the EC50 value is higher than 1000 mg/L. This value was used for risk assessment. The specific respiration rates were compared with the blank control values usingDunnett t-test (a=0.05).Without pH adjustmentthe specific respiration rates did not differ statistically significantly from the blank control inthe concentration range of 3.2 - 320 mg/L,and differed statistically significantly at the concentrations of 640 and 1000 mg/L(Dunnett t-test,a=0.05). Without pH adjustment the NOEC can be statistically and biologically determined as 320 mg/L. With pH adjustmentthe specific respiration rates did not differ statistically significantly from the blank control up and including the concentration of 1000 mg/L(Dunnett t-test,a=0.05). With an additional pH adjustment, neutralization step, the NOEC can be statistically and biologically determined as 1000 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item 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 that the test item caused effect on the activated sludge inoculum, the test item was investigated at the nominal concentrations of 10, 32, 100, 320, 640 and 1000 mg/L. Defined amounts of the test item were added (measured) directly into the test vessels. These test item concentrations were tested without pH adjustment. Based on the preliminary experience, the test item affects adversely the pH within the test system; therefore, a second series of test containers were investigated at the concentrations of 320, 640 and 1000 mg/L. In these additional containers pH adjustment (an additional neutralization step to the required pH range of 7-8) was performed with 1N HCl prior to inoculum addition. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L ran; 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. All validity criteria of the study were met.The pH of the test mixtures (before inoculum addition) remained within the required range of pH 7 – 8 at the concentrations of 10 and 32 mg/L. Without pH adjustment the pH of test mixtures (before inoculum addition) varied between 8.52 - 8.53 at 100 mg/L, between 9.71 - 9.73 at 320 mg/L, between 10.29-10.31 at 640 mg/L and it varied between 10.54 - 10.58 at 1000 mg/L. Inhibition was not noticed in the concentrations of 10 and 32 mg/L concentrations. The obtained 8.75 % inhibition at the concentration of 100 mg/L was evaluated rather as being within the biological variability range of the applied test system, than a test item effect. No inhibition was noticed at 320 mg/L at both conditions: with and without pH setting. Without pH setting 36.34 % inhibition was obtained at 640 mg/L and 76.71 % at 1000 mg/L. No inhibition was noticed in the whole examined concentration range up to 1000 mg/L, with pH setting. The degree of inhibition of oxygen consumption rates (36.34 and 76.71 %) obtained without pH adjustment at the concentration levels of 640 and 1000 mg/L allowed the calculation of the 3-hour exact EC10, EC50 values and estimation of EC80 values. Based on the available data the 3-hour EC10, EC50 and EC80 values and their 95 % confidence limits were calculated (in the case of EC80 estimated) by Probit analysis. The values are the following:

EC10 : 362.5 mg/L    (95 % conf. limits: 33.0–538.8 mg/L),

EC50 : 772.4 mg/L    (95 % conf. limits: 597.8–1073.0 mg/L),

EC80 : 1041.6 mg/L  (95 % conf. limits: 828.8–1563.7 mg/L).

Without pH adjustment, the EC50 value was determined as 772.4mg/L, consequently1000 mg/L > EC50> 100 mg/L.

With additional neutralization step no inhibition was noticed up to 1000 mg/L. In this case the available data did not allow the calculation of exact ECx values.

With additional neutralization step (before the inoculation) the EC50 value is higher than 1000 mg/L. This value was used for risk assessment. The specific respiration rates were compared with the blank control values usingDunnett t-test (a=0.05).Without pH adjustmentthe specific respiration rates did not differ statistically significantly from the blank control inthe concentration range of 3.2 - 320 mg/L,and differed statistically significantly at the concentrations of 640 and 1000 mg/L(Dunnett t-test,a=0.05). Without pH adjustment the NOEC can be statistically and biologically determined as 320 mg/L.With pH adjustmentthe specific respiration rates did not differ statistically significantly from the blank control up and including the concentration of 1000 mg/L(Dunnett t-test,a=0.05).With an additional pH adjustment, neutralization step, the NOEC can be statistically and biologically determined as 1000 mg/L.