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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2018 to 06 June 2018
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))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1 000 mg/L. 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. 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 1 000 mg/L (three replicates at 1 000 mg/L). The pH of the test material dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and, adjusted to between pH 7.0 and 8.0.
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 (7.5 litres) was fed synthetic sewage (375 mL). The pH of the sample on the day of the test was 7.4 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 3 times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed three 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 1-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 prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 - 21 °C
pH:
Adjusted to between pH 7.0 and 8.0.
Dissolved oxygen:
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.
Nominal and measured concentrations:
Nominal: 10, 100 and 1 000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Conical flask
- Type: Closed
- Material, size, headspace, fill volume: Final volume of 500 mL
- Aeration: Yes. The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 litre per 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 range-finding test was between pH 7.2 and 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.

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 using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.

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 1 000 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:
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:
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 1 000 mg/L.
It was not possible to determine an EC50 value and 95 % confidence limits for the test material as no concentration tested resulted in greater than 50 % inhibition.
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.

VALIDATION CRITERIA
- The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 5.2 mg/L.
- The specific respiration rate of the controls was 28.67 mg oxygen per gram dry weight of sludge per hour.
- The coefficient of variation of oxygen uptake in the control vessels was 2.8 %.
All validation criteria for the study were therefore 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 5.2 mg/L with 95 % confidence limits of 3.9 to 7.0 mg/L.
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration for the reference item 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 item were determined by inspection of the inhibition of respiration rate data.
The 95 % confidence limits were calculated for the reference item 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 (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data 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

4.2

3

2.0

44.00

-

R2

4.9

4

2.1

42.00

-

R3

4.5

4

1.7

42.00

-

R4

4.2

3

2.0

44.00

-

Test material

10

4.6

4

1.8

42.00

2

100

4.9

4

2.0

43.50

[1]

1000 R1

4.7

4

2.0

40.50

6

1000 R2

5.0

4

2.2

42.00

2

1000 R3

4.9

4

2.1

42.00

2

Positive control

3.2

6.2

9

2.1

27.33

36

10

6.7

10

3.9

16.80

70

32

7.8

10

7.0

4.80

89

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

- = Not applicable

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1 000 mg/L. The NOEC after 3 hours exposure was 1 000 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 1 000 mg/L (three replicates of the 1 000 mg/L test concentration) for a period of 3 hours at a measured temperature of between 20 °C and 21 °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.

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1 000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1 000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1 000 mg/L.

The reference material, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 5.2 mg/L, 95 % confidence limits 3.9 to 7.0 mg/L confirming the acceptability of the inoculum used in the test system.

Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1 000 mg/L. The NOEC after 3 hours exposure was 1 000 mg/L.

Description of key information

Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1 000 mg/L. The NOEC after 3 hours exposure was 1 000 mg/L.

Key value for chemical safety assessment

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 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 1 000 mg/L (three replicates of the 1 000 mg/L test concentration) for a period of 3 hours at a measured temperature of between 20 °C and 21 °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.

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1 000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1 000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1 000 mg/L.

The reference material, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 5.2 mg/L, 95 % confidence limits 3.9 to 7.0 mg/L confirming the acceptability of the inoculum used in the test system.

Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1 000 mg/L. The NOEC after 3 hours exposure was 1 000 mg/L.