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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-07-2020 to 29-01-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, weighed amounts were added the 1-liter test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis), with initial concentration of 2.5 times the final concentration. These mixtures were stirred in closed dark brown bottles for short period of time. Subsequently, 16 mL synthetic medium, made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: 3,5-dichlorophenol stock solution with a final concentration of 1.0 g/L in Milli-RO water was prepared. The pH as used for the test was 7.7, in the range-finding and definitive tests, respectively. The stock solution were stored in a freezer until use. On the day of testing the reference substance solution was defrosted at room temperature and diluted to reach the test concentrations. The concentrations tested were : 1.0, 3.2, 10 and 32 mg/L. A negative/blank control without test item or reference item was also included.
- 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.): None reported.
- Other: Test designs:
(i) Range-Finding Tests : 1 : The sludge was buffered to a pH of 7.0 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. Three concentrations: 10, 100 and 1000 mg/L (lowest concentration in duplicate ; highest concentration in triplicate and furthermore, three replicates of the highest concentration with nitrification inhibitor and two replicates of a nitrification control). Inhibitor of nitrification : 2.32 g/L solution of N-allylthiourea (ATU) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L). Controls: test medium without test item, treated in the same way as the test item solutions. Abiotic control only test item without sludge. Nitrification control, test medium without test item but with nitrification inhibitor.
(ii) Range-Finding Tests : 2: The pH was 6.9 on the day of testing. No adjustment was required. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. Three concentrations: 10, 100 and 500 mg/L (lowest concentration in duplicate ; highest concentration in triplicate and furthermore, three replicates of the highest concentration with nitrification inhibitor and two replicates of a nitrification control). Inhibitor of nitrification : 2.32 g/L solution of N-allylthiourea (ATU) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L). Controls: test medium without test item, treated in the same way as the test item solutions. Abiotic control only test item without sludge. Nitrification control, test medium without test item but with nitrification inhibitor.
(iii) Definitive Test : 1 : The pH was 7.5 on the day of testing. No adjustment was required. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. Test concentrations: 3.3, 10, 30, 90, 270 and 810 mg/L (5 replicates per test group and 6 replicates in the controls) Inhibitor of nitrification : 2.32 g/L solution of N-allylthiourea (ATU) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L). Controls: test medium without test item, treated in the same way as the test item solutions. Nitrification control, test medium without test item but with nitrification inhibitor.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: aerobic activated sludge from sewage treatment plant at Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, which treats predominantly domestic sewage (dates provided in the full study report).
- Preparation of inoculum for exposure: The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The batch of sludge was used one
day after collection. Therefore 50 mL of synthetic medium was added per liter of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use. The amount of suspended solids in the final test mixture was 1.5 g/L.
- Initial biomass concentration: Determination of the suspended solids level of the activated sewage sludge was carried out. The suspended solids concentration was equal to 3.0 g/L prior to use. The amount of suspended solids in the final test mixture was 1.5 g/L (with and without ATU, nitrification inhibitor).
- Other: Synthetic waste water (synthetic medium) was prepared according to OECD 209. Adjusted ISO-medium, formulated using RO water (tap water
purified by reverse osmosis) with the following composition: CaCl2.2H2O 211.5 mg/L ; MgSO4.7H2O 88.8 mg/L ; NaHCO3 46.7 mg/L ; KCl 4.2 mg/L. The pH value of the activated sludge was determined prior to test start. First range-finding test: the sludge was buffered to a pH of 7.0 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. Second range-finding test: the pH was 6.9 on the day of testing. Adjustment to 7.5 ± 0.5 was not necessary. Definitive test: the pH was 7.5 on the day of testing. Adjustment to 7.5 ± 0.5 was not necessary. In the reference item control : the pH was 7.7 on the day of testing in the first (range finding) and second (definitive) tests, respectively. The batch of sludge was used one day after collection; therefore 50 mL of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use. The synthetic medium / sewage feed (16 mL) and an adequate amount of the test item loading solution (200 mL) were mixed and made up to 250 mL with RO water in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device. After the 3-hour contact time, the oxygen consumption was recorded for a period of 10-11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
In accordance with the OECD TG 209 guideline.
Post exposure observation period:
The synthetic medium (16 mL) and an adequate amount of the test item loading solution (200 mL) were mixed and made up to 250 mL with RO water in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.
Test temperature:
20 ± 2 °C
pH:
See "any other information on materials and methods incl. tables"
Dissolved oxygen:
aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension
Nominal and measured concentrations:
range-finding test 1:
Test item concentrations: 0 (control), 10, 100, 1000 mg/L
range-finding test 2:
Test item concentrations: 0 (control), 10, 100, 500 mg/L
Reference item was completed: nominal: 1.0, 3.2, 10, 32 mg/L (in single replicates)
Along with nitrification control and n-allylthiourea + test item and/or abiotic control (test item) single or three replicates, as applicable.
Definitive Test concentrations:
0 (control), 3.3, 10, 30, 90, 270, 810 mg/L (five replicates and six for control) – without and with ATU (nitrification inhibitor)
Reference item was completed: nominal: 1.0, 3.2, 10, 32 mg/L (in single replicates)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L glass BOD bottle
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start is above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
- 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): Six (negative) and three (reference item)
- Nitrification inhibitor used (delete if not applicable): Testing included nitrification controls and/or nitrification inhibitor and abiotic conditions replicates.
- Biomass loading rate: See table.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC). Synthetic sewage was subsequently prepared.
- Culture medium different from test medium: Not applicable.
- 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. Dissolved oxygen values were determined in all vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Light intensity: The test was conducted under normal laboratory lighting ; glassware was wrapped in aluminium foil and preparation of test solutions was performed under dimmed light conditions to minimize exposure to light.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological Oxygen Demand. Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations:
range-finding test 1:
Test item concentrations: 0 (control), 10, 100, 1000 mg test item/L
range-finding test 2:
Test item concentrations: 0 (control), 10, 100, 500 mg test item/L
Reference item was completed: nominal: 1.0, 3.2, 10, 32 mg/L (in single replicates)
Along with nitrification control and n-allylthiourea + test item and/or abiotic control (test item) single or three replicates, as applicable.
Definitive Test concentrations:
0 (control), 3.3, 10, 30, 90, 270, 810 mg/L (five replicates and six for control) – without and with ATU (nitrification inhibitor)
Reference item was completed: nominal: 1.0, 3.2, 10, 32 mg/L (in single replicates)
- Range finding study: See above
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 810 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
C.I. - mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
270 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: no statistically significant inhibition was recorded up to 270 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 270 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other:
Remarks:
C.I. - mg/
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
270 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 was recorded up to 270 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 270 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: no statistically significant inhibition was recorded at any of the concentrations tested.
Remarks:
C.I. - mg/
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
810 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: no statistically significant inhibition was recorded at any of the concentrations tested.
Details on results:
Validity Criteria:
1. The controls oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. (Actual = 25 mgO2/h)
2. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test. (Actual = 10%).
3. The reference substance results were valid, total respiration: the EC50 for 3,5-dichlorophenol was: actual 9.0 mg/L. This was within the expected range: 2 to 25 mg/L ; heterotrophic respiration : actual 29.5 mg/L. This was within the expected range: 2 to 40 mg/L ; nitrification : the EC50 for 3,5-dichlorophenol was: actual 1.0 mg/L. This was within the expected range: 2 to 10 mg/L. Full information is provided in the full study report.
Therefore, the validity criteria was met.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: total respiration: the EC50 for 3,5-dichlorophenol was: actual 9.0 mg/L. This was within the expected range: 2 to 25 mg/L ; heterotrophic respiration : actual 29.5 mg/L. This was within the expected range: 2 to 40 mg/L ; nitrification : the EC50 for 3,5-dichlorophenol was: actual 1.0 mg/L. This was within the expected range: 2 to 10 mg/L. Full information is provided in the full study report.
Reported statistics and error estimates:
95% confidence limits were calculated for the reference item EC50 value using linear regression analysis.

Table 1.0 – Study results, First Range-Finding Test – Respiration Rate/Inhibition, pH Values :

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

 

Start

End

(mg O2/L h)

(mg O2/g h) #1

 

C 1

0

7.6

7.7

23.62

15.75

 

 

C 2

0

7.6

7.7

26.01

17.34

 

 

C 3

0

7.6

7.7

25.48

16.99

 

 

C 4

0

7.6

7.7

31.03

20.69

 

 

C 5

0

7.6

7.7

35.68

23.79

 

 

C 6

0

7.6

7.5

#2

#2

 

 

C Mean

 

 

 

28.36

18.91 (RTB)

 

 

SD

 

 

 

4.92

3.28

 

 

CV (%)

 

 

 

17

17

 

 

 

 

 

 

 

 

 

CN 1

0

7.6

8.2

11.76

7.84

 

 

CN 2

0

7.6

8.2

15.90

10.60

 

 

CN Mean

 

 

13.83

9.22 (RHB)

 

 

 

 

 

 

 

 

 

 

R 1

1.0

7.6

8.0

18.77

12.51

 

 

R 2

3.2

7.6

8.0

16.58

11.05

 

 

R 3

10

7.6

7.9

2

2

 

 

R 4

32

7.6

8.0

2.22

1.48

 

 

 

 

 

 

 

 

 

 

T 1

10

7.4

7.8

25.78

17.19

9.11

 

T 2

100

6.4

7.5

29.72

19.81

-4.78

 

T 3a

1000

2.5

5.0

2.69

1.79

90.52

 

T 3b

1000

2.5

5.0

#2

#2

#2

 

T 3c

1000

2.5

5.0

1.18

0.79

95.84

 

T3 Mean

 

 

1.94

0.71 (RT)

93.18 (IT)

 

 

 

 

 

 

 

 

 

TN a

1000

2.5

5.1

2.44

1.63

82.36

 

TN b

1000

2.5

5.0

#2

#2

#2

 

TN c

1000

2.5

5.0

1.10

0.73

92.05

 

TN Mean

 

 

1.77

1.18 (RH)

87.20 (IH­)

 

 

 

 

 

 

 

 

 

TA

1000

2.5

2.8

0.00#

 

 

 

C:

Blank control

#1

The amount of suspended solids in the final test mixture was 1.5 g/L

CN:

Nitrification control

#2:

Unreliable measurement due to a deviating respiration curve, therefore this measurement was excluded

R:

Reference item, 3,5-dichlorophenol

RTB:

Total respiration in the blank

T:

Test item

RHB:

Heterotrophic respiration in the nitrification control

TA:

Abiotic control of Test Item

RT:

Total respiration with Test Item

TN:

Test Item with N-allylthiourea

RH:

Heterotrophic respiration with Test Item

SD:

Standard deviation

IT:

% inhibition of total respiration relative to RTB

CV:

Coefficient of variation

IH:

% inhibition of heterotrophic respiration relative to RHB

#

No respiration, therefore expressed as 0 mg O2/L h

 

 


Table 2.0 – Study results, Second Range-Finding Test – Respiration Rate/Inhibition, pH Values :

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

 

Start

End

(mg O2/L h)

(mg O2/g h) #1

 

C 1

0

7.4

7.2

30.04

20.03

 

 

C 2

0

7.4

7.3

24.52

16.35

 

 

C 3

0

7.4

7.2

29.63

19.75

 

 

C 4

0

7.4

7.2

29.35

19.57

 

 

C 5

0

7.4

7.1

32.19

21.46

 

 

C 6

0

7.4

7.1

28.66

19.11

 

 

C Mean

 

 

 

29.07

19.38 (RTB)

 

 

SD

 

 

 

2.53

1.68

 

 

CV (%)

 

 

 

9

9

 

 

 

 

 

 

 

 

 

CN 1

0

7.4

7.7

18.30

12.20

 

 

CN 2

0

7.4

7.7

19.53

13.02

 

 

CN Mean

 

 

18.92

12.61 (RHB)

 

 

 

 

 

 

 

 

 

 

R 1

1.0

7.4

7.6

21.04

14.03

27.61

 

R 2

3.2

7.4

7.6

16.67

11.11

42.65

 

R 3

10

7.4

7.5

8.97

5.98

69.14

 

R 4

32

7.4

7.4

1.68

1.12

94.22

 

 

 

 

 

 

 

 

 

T 1

10

7.3

7.3

22.13

14.75

23.86

 

T 2

100

6.3

6.9

28.44

18.96

2.15

 

T 3a

500

3.7

5.3

3.02

2.01

89.61

 

T 3b

500

3.8

5.5

2.43

1.62

91.64

 

T 3c

500

3.6

5.3

2.87

1.91

90.13

 

T3 Mean

 

 

2.77

1.85 (RT)

90.46 (IT)

 

 

 

 

 

 

 

 

 

TN a

500

2.3

5.3

2.21

1.47

88.32

 

TN b

500

2.3

5.3

3.16

2.11

83.29

 

TN c

500

2.3

5.3

3.10

2.07

83.61

 

TN Mean

 

 

2.82

1.88 (RH)

85.07

 

 

 

 

 

 

 

 

 

TA

500

3.6

3.7

0.00#

 

 

 

C:

Blank control

#1

The amount of suspended solids in the final test mixture was 1.5 g/L.

CN:

Nitrification control

RTB:

Total respiration in the blank

R:

Reference item, 3,5-dichlorophenol

RHB:

Heterotrophic respiration in the nitrification control

T:

Test item

RT:

Total respiration with Test Item

TA:

Abiotic control of Test Item

RH:

Heterotrophic respiration with Test Item

TN:

Test Item with N-allylthiourea

IT:

% inhibition of total respiration relative to RTB

SD:

Standard deviation

IH:

% inhibition of heterotrophic respiration relative to RHB

CV:

Coefficient of variation

 

 

#

No respiration, therefore expressed as 0 mg O2/L h

 

 


Table 3.0 – Study results, Final Test – Respiration Rate/Inhibition :

Concentration

(mg/L)

Mean respiration rate

% Inhibition of the respiration rate
(mean value)

(mg O2/L h)

(mg O2/g h) #1

Total respiration (T)

0

37.83

25.22

 

3.3

37.92

25.28

-0.2

10

41.92

27.95

-11

30

43.36

28.91

-15

90

47.42

31.61

-25

270

41.11

27.41

-8.7

810

17.47

11.64 *

54

Heterotrophic respiration (H)

0

30.93

20.62

 

3.3

33.87

22.58

-9.5

10

40.48

26.99

-31

30

40.16

26.77

-30

90

32.68

21.79

-5.6

270

32.61

21.74

-5.4

810

11.00

7.33 *

64

Nitrification respiration (N) #2

0

6.90

4.60

 

3.3

4.05

2.70

41

10

1.44

0.96

79

30

3.20

2.13

54

90

14.74

9.82

-114

270

8.50

5.67

-23

810

6.47

4.31

6.2

#1 : The amount of suspended solids in the final test mixture was 1.5 g/L.

#2 : Coefficient of variation (CV) of nitrification respiration rate in control replicates was 54%

* Statistically significantly different compared to control.

The pH in all test vessels, before addition of sludge, was between 2.6 and 7.2. After the 3 hour exposure period the pH was between 6.3 and 8.3.

 

Complete study results (including Respiration Rate/Inhibition, pH Values, and statistical analyses output) are available in the full study report.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3-hour EC50 for total respiration inhibition and heterotrophic respiration inhibition was > 270 (C.I. - ) mg/L. The equivalent NOEC was 270 mg/L. There was no specific inhibition of nitrification observed within the study. NOEC nitrification respiration inhibition was > 810 mg/L. All effect levels were based on nominal test item concentrations.
Executive summary:

The effect on respiration rate of activate sludge of the test item was examined using OECD TG 209 in accordance with GLP. The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, weighed amounts were added the 1-liter test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis), with initial concentration of 2.5 times the final concentration. These mixtures were stirred in closed dark brown bottles for short period of time. Subsequently, 16 mL synthetic medium, made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Two independent range-finding tests were conducted (in 1: the sludge was buffered to a pH of 7.0 using a 70 g/L sodium bicarbonate solution and in 2: no sludge pH adjustment was required) with and without N-allylthiourea (ATU) nitrification inhibition along with appropriate blank and/or reference item (3,5-dichlorophenol) and/or abiotic controls. Subsequently, a definitive test was conducted. Within this the pH of the sludge was 7.5 on the day of testing. No adjustment was required. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. Definitive test concentrations of test item were : 3.3, 10, 30, 90, 270 and 810 mg/L (5 replicates per test group and 6 replicates in the controls). For the inhibitor of nitrification : 2.32 g/L solution of N-allylthiourea (ATU) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L). For controls: test medium without test item, treated in the same way as the test item solutions. Six replicates of blank controls and three replicates of reference item controls along with appropriate nitrification controls were included. All validity criteria were met. The controls oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test. The reference substance results were valid, total respiration:  the EC50 for 3,5-dichlorophenol was: actual 9.0 mg/L. This was within the expected range: 2 to 25 mg/L ; heterotrophic respiration : actual 29.5 mg/L. This was within the expected range: 2 to 40 mg/L ; nitrification : the EC50 for 3,5-dichlorophenol was: actual 1.0 mg/L. This was within the expected range: 2 to 10 mg/L. For total respiration rate: no statistically significant inhibition was recorded up to 270 mg/L; a statistically significant inhibition of 54% was observed at the highest concentration. For heterotrophic respiration rate: no statistically significant inhibition was recorded up to 270 mg/L; a statistically significant inhibition of 64% was observed at the highest concentration. For nitrification respiration rate: no statistically significant inhibition was recorded at any of the concentrations tested. Under the conditions of the study, the 3-hour EC50 for total respiration inhibition and heterotrophic respiration inhibition was > 270 (C.I. - ) mg/L. The equivalent NOEC was 270 mg/L. There was no specific inhibition of nitrification observed within the study. NOEC nitrification respiration inhibition was > 810 mg/L. All effect levels were based on nominal test item concentrations.

Description of key information

Total Respiration Inhibition:

3h-EC50 = > 270 mg/L (nominal), 20 °C

3h-EC10 = - mg/L (nominal), 20°C

NOEC = 270 mg/L(nominal), 20 °C, OECD TG 209, 2021

 

Heterotrophic Respiration Inhibition:

3h-EC50 = > 270 mg/L (nominal), 20 °C

3h-EC10 = - mg/L (nominal), 20 °C

NOEC = 270 mg/L(nominal), 20 °C, OECD TG 209, 2021

 

Nitrification Inhibition:

3h-EC50 = > 810 mg/L (nominal), 20 °C

3h-EC10 = - mg/L (nominal), 20 °C

NOEC = 810 mg/L(nominal), 20 °C, OECD TG 209, 2021

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
270 mg/L

Additional information

Key study : OECD TG 209, 2021 : The effect on respiration rate of activate sludge of the test item was examined using OECD TG 209 in accordance with GLP. The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, weighed amounts were added the 1-liter test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis), with initial concentration of 2.5 times the final concentration. These mixtures were stirred in closed dark brown bottles for short period of time. Subsequently, 16 mL synthetic medium, made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Two independent range-finding tests were conducted (in 1: the sludge was buffered to a pH of 7.0 using a 70 g/L sodium bicarbonate solution and in 2: no sludge pH adjustment was required) with and without N-allylthiourea (ATU) nitrification inhibition along with appropriate blank and/or reference item (3,5-dichlorophenol) and/or abiotic controls. Subsequently, a definitive test was conducted. Within this the pH of the sludge was 7.5 on the day of testing. No adjustment was required. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. Definitive test concentrations of test item were : 3.3, 10, 30, 90, 270 and 810 mg/L (5 replicates per test group and 6 replicates in the controls). For the inhibitor of nitrification : 2.32 g/L solution of N-allylthiourea (ATU) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L). For controls: test medium without test item, treated in the same way as the test item solutions. Six replicates of blank controls and three replicates of reference item controls along with appropriate nitrification controls were included. All validity criteria were met. The controls oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test. The reference substance results were valid, total respiration:  the EC50 for 3,5-dichlorophenol was: actual 9.0 mg/L. This was within the expected range: 2 to 25 mg/L ; heterotrophic respiration : actual 29.5 mg/L. This was within the expected range: 2 to 40 mg/L ; nitrification : the EC50 for 3,5-dichlorophenol was: actual 1.0 mg/L. This was within the expected range: 2 to 10 mg/L. For total respiration rate: no statistically significant inhibition was recorded up to 270 mg/L; a statistically significant inhibition of 54% was observed at the highest concentration. For heterotrophic respiration rate: no statistically significant inhibition was recorded up to 270 mg/L; a statistically significant inhibition of 64% was observed at the highest concentration. For nitrification respiration rate: no statistically significant inhibition was recorded at any of the concentrations tested. Under the conditions of the study, the 3-hour EC50 for total respiration inhibition and heterotrophic respiration inhibition was > 270 (C.I. - ) mg/L. The equivalent NOEC was 270 mg/L. There was no specific inhibition of nitrification observed within the study. NOEC nitrification respiration inhibition was > 810 mg/L. All effect levels were based on nominal test item concentrations.