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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 August 2016 to 12 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: Read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.7. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper (rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Initial biomass concentration: The suspended solids concentration was equal to 3.0 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 to 21 °C
pH:
7.8 to 8.0 after adjustment
Dissolved oxygen:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70 % of the dissolved oxygen saturation level of 8.9 mg O2/L.
Nominal and measured concentrations:
- Nominal concentrations: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Type: closed
- Material, size, headspace, fill volume: Final volume of 500 mL
- Aeration: Yes; at the time of preparation of each flask, the mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 L/minute.
- No. of vessels per concentration (replicates): 3 (top dose) and 2 (low and intermediate dose)
- No. of vessels per control (replicates): 4
- No. of vessels per positive control (replicates): 1 per concentration level (3.2, 10 and 32 mg/L)
- Sludge concentration (weight of dry solids per volume): 3.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised reverse osmosis water containing less than 1 mg/L dissolved organic carbon (DOC)
- Synthetic sewage: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 dissolved in a final volume of 1 litre of water with the aid of ultrasonication. The pH of the synthetic sewage stock was 7.3.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, adjusted to between pH 7 and 8 if necessary
- Photoperiod: Continuous
- Light intensity: Normal laboratory lighting
- Details on termination of incubation: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-hour incubation period. The oxygen concentrations in all vessels were measured after 30 minutes contact time.
The rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 and 2 mg O2/L). In the case of rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
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
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials after 3 hours contact time are given in Table 1.
No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

VALIDATION CRITERIA
The results of the study are considered valid if:
- the EC50 (3-hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L for total respiration.
- the specific respiration rate of the blank controls is not less than 20 mg oxygen per gram dry weight of sludge per hour for total respiration.
- the coefficient of variation of oxygen uptake rate in control replicates is not more than 30 % at the end of the test.
The coefficient of variation of oxygen uptake in the control vessels was 1.75 % and the specific respiration rate of the controls was 24.37 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. The validation criterion for the reference item EC50 value was also satisfied.
Results with reference substance (positive control):
The validation criterion for the reference item EC50 value was satisfied in this study. The EC50 was determined to be 8.4 mg/L with 95 % confidence limits of 6.7 to 11 mg/L.
Reported statistics and error estimates:
ECX and NOEC
The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line. The EC10, EC20, EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data. 95 % confidence limits were calculated for the reference material EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1 Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours of Contact Time

Nominal Concentration (mg/L)

Initial O2 Reading (mg O2/L)

Measurement Period (minutes)

Final O2 Reading (mg O2/L)

O2 Consumption Rates (mg O2/L/hour)

% Inhibition

Control

R1

4.3

4

1.9

36.00

-

R2

5.0

5

1.9

37.20

-

R3

5.2

5

2.2

36.00

-

R4

5.6

6

1.9

37.00

-

Test material

10

4.2

4

1.6

39.00

[7]

100

4.0

3

2.1

38.00

[4]

1000 R1

4.5

4

2.0

37.50

[3]

1000 R2

5.2

6

1.6

36.00

2

1000 R3

4.2

4

1.8

36.00

2

Positive control

3.2

5.7

8

1.8

29.25

20

10

7.1

10

4.5

15.60

57

32

8.1

10

7.4

4.20

89

[ ] = Increase in respiration rate as compared to the controls

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study the 3-hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) was 1000 mg/L.
Executive summary:

The toxicity of the test material to activated sludge was determined in accordance with the standardised guideline OECD 209 under GLP conditions using a respiration inhibition test.

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at temperatures of between 20 and 21 °C with the addition of synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours of contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-hour EC50 value of 8.4 mg/L with 95 % confidence limits of 6.7 to 11 mg/L.

Under the conditions of this study the 3-hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) was 1000 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a reliability 1 study
Reason / purpose for cross-reference:
read-across source
Key result
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
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
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials after 3 hours contact time are given in Table 1.
No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

VALIDATION CRITERIA
The results of the study are considered valid if:
- the EC50 (3-hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L for total respiration.
- the specific respiration rate of the blank controls is not less than 20 mg oxygen per gram dry weight of sludge per hour for total respiration.
- the coefficient of variation of oxygen uptake rate in control replicates is not more than 30 % at the end of the test.
The coefficient of variation of oxygen uptake in the control vessels was 1.75 % and the specific respiration rate of the controls was 24.37 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. The validation criterion for the reference item EC50 value was also satisfied.
Results with reference substance (positive control):
The validation criterion for the reference item EC50 value was satisfied in this study. The EC50 was determined to be 8.4 mg/L with 95 % confidence limits of 6.7 to 11 mg/L.
Reported statistics and error estimates:
ECX and NOEC
The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line. The EC10, EC20, EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data. 95 % confidence limits were calculated for the reference material EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1 Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours of Contact Time

Nominal Concentration (mg/L)

Initial O2 Reading (mg O2/L)

Measurement Period (minutes)

Final O2 Reading (mg O2/L)

O2 Consumption Rates (mg O2/L/hour)

% Inhibition

Control

R1

4.3

4

1.9

36.00

-

R2

5.0

5

1.9

37.20

-

R3

5.2

5

2.2

36.00

-

R4

5.6

6

1.9

37.00

-

Test material

10

4.2

4

1.6

39.00

[7]

100

4.0

3

2.1

38.00

[4]

1000 R1

4.5

4

2.0

37.50

[3]

1000 R2

5.2

6

1.6

36.00

2

1000 R3

4.2

4

1.8

36.00

2

Positive control

3.2

5.7

8

1.8

29.25

20

10

7.1

10

4.5

15.60

57

32

8.1

10

7.4

4.20

89

[ ] = Increase in respiration rate as compared to the controls

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study the 3-hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) was 1000 mg/L.
Executive summary:

The toxicity of the test material to activated sludge was determined in accordance with the standardised guideline OECD 209 under GLP conditions using a respiration inhibition test.

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at temperatures of between 20 and 21 °C with the addition of synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours of contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-hour EC50 value of 8.4 mg/L with 95 % confidence limits of 6.7 to 11 mg/L.

Under the conditions of this study the 3-hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) was 1000 mg/L.

Description of key information

Read-across to structurally similar substance:

Under the conditions of this study the 3-hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

Read-across to structurally similar substance:

The toxicity of the test material to activated sludge was determined in accordance with the standardised guideline OECD 209 under GLP conditions using a respiration inhibition test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at temperatures of between 20 and 21 °C with the addition of synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours of contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-hour EC50 value of 8.4 mg/L with 95 % confidence limits of 6.7 to 11 mg/L.

Under the conditions of this study the 3-hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) was 1000 mg/L.