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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-10-2013 to 17-01-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according 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 completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis), with initial loading rates of 2.5 the final loading rate. These mixtures were stirred in closed dark brown bottles for 5 to 8 minutes. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured 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 g/L in Milli-RO water was prepared. The pH as used for the test was 7.6 and 7.8, in the first and second combined limit/range-finding, respectively. Aliquots of 25 mL 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. Three concentrations were tested: 5.0, 12 and 30 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.
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).
- 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.
- Other: Synthetic waste water was prepared according to OECD 209. The pH value of the activated sludge was determined prior to test start. The pH was 7.6 and 7.7 on the day of testing in the first and second 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
During 3 hour exposure aeration and stirring took place.
Post exposure observation period:
The synthetic sewage feed (16 mL) and an adequate amount of the test substance loading 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 temperature:
20 ± 2 °C
pH:
first combined limit/range-finding test
0 hours: control: 7.6 and test groups: 7.6 and reference item: 7.6 and nitrification control: 7.6 and n-allylthiourea + test item (nitrification inhibitor vessel): 7.6
3 hours: control: 7.8 and test groups: 7.7 – 7.8 and reference item: 8.1 and nitrification control: 8.2 and n-allylthiourea + test item (nitrification inhibitor vessel): 8.1

second combined limit/range-finding test (nitrification control and n-allylthiourea only):
0 hours: control: 6.3 – 7.2 and n-allylthiourea + test item (nitrification inhibitor vessel): 7.4 – 7.5
3 hours: control: 7.5 – 7.7 and n-allylthiourea + test item (nitrification inhibitor vessel): 8.0 – 8.2
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:
first combined limit/range-finding test
Definitive Test concentrations: 10, 100, 1000 mg/L (in single vessels, three replicates for 1000 mg/L and six for control).
Reference item was completed: nominal: 5, 12, 30 mg/L (in single replicates)
Along with nitrification control and n-allylthiourea + test item and/or abiotic control (test item) single replicates

second combined limit/range-finding test (nitrification control and n-allylthiourea only):
Reference item was completed: nominal: 5, 12, 30 mg/L (in single replicates)
Along with nitrification control (six replicates) and n-allylthiourea + test item and/or abiotic control (test item) 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

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: first combined limit/range-finding test
Definitive Test concentrations: 10, 100, 1000 mg/L (in single vessels, three replicates for 1000 mg/L and six for control).
Reference item was completed: nominal: 5, 12, 30 mg/L (in single replicates)
Along with nitrification control and n-allylthiourea + test item and/or abiotic control (test item) single replicates
second combined limit/range-finding test (nitrification control and n-allylthiourea only):
Reference item was completed: nominal: 5, 12, 30 mg/L (in single replicates)
Along with nitrification control (six replicates) and n-allylthiourea + test item and/or abiotic control (test item) single replicates
- Range finding study: Not applicable.
- Results used to determine the conditions for the definitive study: Combined limit/range-finding test was conducted.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: based on two combined/limit-range finding tests (n=2)
Remarks:
C.I. - mg/L
Duration:
3 h
Dose descriptor:
NOEC
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
not determinable
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.
2. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.
3. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: first combined limit/range finding test: total respiration: actual 6.0 (C.I. 1.8 – 19.6) mg/L. This was within the expected range: 2 to 25 mg/L ; second combined limit/range finding test: total respiration: actual 6.6 mg/L. This was within the expected range: 2 to 25 mg/L
Therefore, the validity criteria was met.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was:first combined limit/range finding test: total respiration: actual 6.0 (C.I. 1.8 – 19.6) mg/L. This was within the expected range: 2 to 25 mg/L ; second combined limit/range finding test: total respiration: actual 6.6 mg/L. This was within the expected range: 2 to 25 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 combined limit/range-finding test:

 

 

 

Flask

 

 

Loading rate (mg/l)

 

 

Temp. (°C)

 

 

pH before addition of sludge

 

 

pH after 3 h contact time

 

 

Respiration rate

(mg O2/l h)

 

 

Respiration rate

(mg O2/g h)¹

 

% Inhibition respiration relative to the control

(mean value)

C 1

0

20.4

7.6

7.8

32

21

 

C 2

0

20.9

7.6

7.8

32

21

 

C 3

0

20.1

7.6

7.8

34

23

 

C 4

0

20.8

7.6

7.8

36

24

 

C 5

0

20.7

7.6

7.8

34

23

 

C 6

0

20.6

7.6

7.8

30

20

 

C Mean

 

 

 

 

33 (=RTB)

22

 

SD

 

 

 

 

2

1

 

CV (%)

 

 

 

 

6

6

 

 

 

 

 

 

 

 

 

CN

 

20.2

7.6

8.2

#

#

#

 

 

 

 

 

 

 

 

R 1

5.0

20.7

7.6

8.1

18

12

45

R 2

12

20.6

7.6

8.1

11

7

67

R 3

30

20.2

7.6

8.1

5

3

85

 

 

 

 

 

 

 

 

T 1

10

20.8

7.6

7.8

39

26

-18

T 2

100

20.8

7.6

7.7

31

21

6

T 3a

1000

20.1

7.6

7.8

18

12

45

T 3b

1000

21.4

7.6

7.7

21

14

36

T 3c

1000

21.8

7.6

7.7

21

14

36

T 3 Mean

 

 

 

 

20 (=RT)

13

39* (=IT)

 

 

 

 

 

 

 

 

TA

1000

20.2

7.6

7.4

0

0

100

TN

1000

21.7

7.6

8.1

#

#

#

C:           Blank control

CN:        Nitrification control

R:           Reference substance, 3,5-dichlorophenol

T:           Test item

SD:        Standard deviation

CV:        Coefficient of variation

TA:        Abiotic control of test item

TN:        Test item with N-allylthiourea.

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

*            Statistically significant different compared to C (Two sample t-Test: α=0.05)

#            These values were considered unreliable (repeated ; see below).

 

Table 2.0 – Study results, second combined limit/range-finding test:

 

 

 

Flask

 

 

Loading rate (mg/l)

 

 

pH before addition of sludge

 

 

 

pH after 3 h contact time

 

 

 

Respiration rate (mg O2/l.h)

 

 

Respiration rate

(mg O2/g.h)¹

% Inhibition

respiration relative to

the blank control (mean value)

C 1

0

7.5

7.6

61

41

 

C 2

0

7.5

7.6

53

35

 

C 3

0

7.5

7.8

53

35

 

C 4

0

7.5

7.6

53

35

 

C 5

0

7.5

7.6

55

37

 

C 6

0

7.5

7.7

47

31

 

 

C Mean

 

 

 

 

54 (=RTB)

 

36

 

SD

 

 

 

5

3

 

CV (%)

 

 

 

8

8

 

 

 

 

 

 

 

 

CN 1

0

7.5

8.1

27

18

50

CN 2

0

7.5

8.1

27

18

50

CN Mean

= RHB

 

 

 

 

27 (=RHB)

 

18

 

 

 

 

 

 

 

 

R 1

5.0

7.5

8.0

31

21

42

R 2

12

7.5

8.0

18

12

66

R 3

30

7.5

8.1

9

6

83

 

 

 

 

 

 

 

T 1

10

7.2

7.6

60

40

-12

T 2

100

7.2

7.5

37

25

31

T 3a

1000

6.8

7.7

26

17

52

T 3b

1000

6.5

7.6

25

17

53

T 3c

1000

6.3

7.5

24

16

55

 

T3 Mean

 

25 (=RT)

 

17

 

53* (=IT)

TN 1

7.5

8.0

16

11

41

TN 2

7.4

8.2

10

7

63

TN 3

7.5

8.1

13

9

52

 

TN Mean

 

13 (=RH)

 

9

 

522 (=IH)

TA

1000

5.9

7.3

0

0

100

C:           Blank control

CN:        Nitrification control

R:           Reference substance, 3,5-dichlorophenol

SD:        Standard deviation

CV:        Coefficient of variation

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

T:           Test item

TA:        Abiotic control of test item

TN:        Test item with N-allylthiourea.

2             Relative to CN

*            Statistically significant different compared to C (Two sample t-Test: α=0.05)

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3-hour EC50 for total inhibition was ca. 1000 (C.I. - ) mg/L. Nitrification and heterotrophic inhibition rates were considered to be comparable to the total inhibition at ca. 1000 mg/L. The NOEC with no significant inhibition of respiration rates could not be determined under the conditions of the study. All effect levels were based on nominal test item concentrations.
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to EU Method C.11, OECD TG 209 and ISO 8192 (2007) in accordance with GLP. The test item was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 5 minutes. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring. Thereafter, oxygen consumption was recorded for 10 to 11 minutes. In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank and nitrification controls of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested. Responses were compared to the blank and nitrification control. During the first combined limit/range-finding test the results for the nitrification control and the highest concentration with a nitrification inhibitor were considered unreliable. Therefore, a second experiment was performed. In both the combined limit/range-finding tests statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg test item per litre (Two Sample t-Test: α=0.05). Therefore, a NOEC could not be determined. At 1000 mg/L an average inhibition of 39% and 53% was observed, in the first and second combined limit/range-finding test, respectively. The nitrification and heterotrophic inhibition was comparable to the total inhibition (55 and 52%, respectively). Based on both experiments, the 3h-EC50 for total inhibition was estimated to be approximately at the highest loading rate tested (1000 mg/L). There was no oxygen uptake from abiotic processes. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid. Under the conditions of the study, the 3-hour EC50 for total inhibition was ca. 1000 (C.I. - ) mg/L. Nitrification and heterotrophic inhibition rates were considered to be comparable to the total inhibition at ca. 1000 mg/L. The NOEC with no significant inhibition of respiration rates could not be determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

Description of key information

ASRIT: EC50-3h = 1000 (C.I. - ) mg/L (nominal), 20 °C, OECD TG 209, 2014

ASRIT: EC20-3h = - (C.I. - ) mg/L (nominal), 20°C, OECD TG 209, 2014

ASRIT: EC10-3h = - (C.I. - ) mg/L (nominal), 20°C, OECD TG 209, 2014

ASRIT: NOEC-3h = - mg/L (nominal), 20 °C, OECD TG 209, 2014

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Key study : OECD TG 209, 2014 : The effect on respiration rate of activate sludge was examined using a method according to EU Method C.11, OECD TG 209 and ISO 8192 (2007) in accordance with GLP. The test item was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 5 minutes. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring. Thereafter, oxygen consumption was recorded for 10 to 11 minutes. In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank and nitrification controls of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested. Responses were compared to the blank and nitrification control. During the first combined limit/range-finding test the results for the nitrification control and the highest concentration with a nitrification inhibitor were considered unreliable. Therefore, a second experiment was performed. In both the combined limit/range-finding tests statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg test item per litre (Two Sample t-Test: α=0.05). Therefore, a NOEC could not be determined. At 1000 mg/L an average inhibition of 39% and 53% was observed, in the first and second combined limit/range-finding test, respectively. The nitrification and heterotrophic inhibition was comparable to the total inhibition (55 and 52%, respectively). Based on both experiments, the 3h-EC50 for total inhibition was estimated to be approximately at the highest loading rate tested (1000 mg/L). There was no oxygen uptake from abiotic processes. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid. Under the conditions of the study, the 3-hour EC50 for total inhibition was ca. 1000 (C.I. - ) mg/L. Nitrification and heterotrophic inhibition rates were considered to be comparable to the total inhibition at ca. 1000 mg/L. The NOEC with no significant inhibition of respiration rates could not be determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

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