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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 November 2017 to 07 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Details on test solutions:
- The test material was dispersed directly in water.
- Nominal amounts of test material (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing.
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 overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.5 measured using a Hach HQ40d Flexi handheld meter.
- 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 deionised 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 °C
pH:
7.1-7.8
Dissolved oxygen:
7.07-8.92 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 1 (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 total organic carbon (TOC)
- 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 used to feed the activated sewage sludge and used in the test was 7.3.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, adjusted to between pH 7.0 and 8.0 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. 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 mg O2/L and 2 mg O2/L). In the case of a 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.

EFFECT PARAMETERS MEASURED:
- 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 using a Hach HQ40d Flexi handheld meter.
- The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- The rate of respiration was 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
- In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test material was dispersed directly in water.
- Nominal amounts of test material (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
- At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated and thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference material vessels with appropriate amounts of the reference material being added. The test material vessels were prepared and finally two further control vessels were prepared.
- 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:
EC50
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:
NOEC
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.
- The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L.
- 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.
- In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
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 5.6 mg/L with 95 % confidence limits of 4.0 to 7.8 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).

Validation Criteria

- The coefficient of variation of oxygen uptake in the control vessels was 1.5 % and the specific respiration rate of the controls was 23.47 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.

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

5.4

6

1.9

35.00

-

R2

5.7

6

2.2

35.00

-

R3

5.5

5

2.5

36.00

-

R4

4.9

5

2.0

34.80

-

Test material

10

5.3

6

1.8

35.00

1

100

5.5

6

1.9

36.00

[2]

1000 R1

5.6

6

2.1

35.00

1

1000 R2

5.7

6

2.2

35.00

1

1000 R3

4.7

5

1.7

36.00

[2]

Positive Control

3.2

6.9

10

3.4

21.00

40

10

7.5

10

5.1

14.40

59

32

8.0

10

7.2

4.80

86

[ ] = 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 an activated sludge 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 measured temperatures of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours 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 5.6 mg/L, 95 % confidence limits 4.0 to 7.8 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

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

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

Additional information

The toxicity of the test material to activated sludge was determined in accordance with the standardised guideline OECD 209 under GLP conditions using an activated sludge 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 measured temperatures of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours 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 5.6 mg/L, 95 % confidence limits 4.0 to 7.8 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.