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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 December 2015 to 13 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Evaluations of appearance, pH measurements, oxygen concentration, and measurement of the respiration rates were performed although no instruction was given to do them. These deviations to the study plan were considered to have had no adverse on the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Physical state: Yellow semi-solid
- Analytical purity: 100 %
- Expiration date of the lot/batch: 23 November 2017
- Storage condition of test material: Room temperature in the dark
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
RANGE FINDING TEST
-Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L.
- Test item was dispersed directly in water.
- Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water.
- Ultrasonication was applied for approximately 15 minutes followed by magnetic stirring for 24 hours at temperatures between 20 °C and 21 °C to maximise the concentration of the dissolved test item.
- All test vessels were shielded from 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).
- The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter (see Table 1, attached) and adjusted to between pH 7.0 and pH 8.0.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- Based on the range-finding test, test concentrations of 56, 100 and 180 mg/L were assigned to the definitive test.
- Nominal amounts of test item (28, 50 and 90 mg (5 replicates of each)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water.
- Ultrasonication was applied for approximately 15 minutes followed by magnetic stirring for 24 hours at temperatures between 20 °C and 21 °C to maximise the concentration of the dissolved test item.
- All test vessels were shielded from 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 56, 100 and 180 mg/L (5 replicates).
- The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter (see Table 6, attached) and adjusted to between pH 7.0 and pH 8.0.
- As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity concentrations or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
- The control group was maintained under identical conditions but not exposed to the test item.

ADDITIONAL EXPERIMENT
-An additional experiment was performed to confirm the NOEC determined from the definitive test.
- A nominal amount of test item (160 mg (5 replicates)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water.
- Ultrasonication was applied for approximately 15 minutes followed by magnetic stirring for 24 hours at temperatures between 20 °C and 21 °C to maximise the concentration of the dissolved test item.
- All test vessels were shielded from 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 320 mg/L (5 replicates).
- The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter (see Table 11, attached) and adjusted to between pH 7.0 and pH 8.0.
- The control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
- A mixed population of activated sewage sludge microorganisms was obtained on 17 December 2015 for the range-finding rest, on 4 January 2016 for the definitive test and on the 12 January 2016 for the additional experiment from the aeration stage of the Severn Trent Water plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

SYNTHETIC SEWAGE
- Synthetic sewage of the following composition was added to each test vessel to act as a respiratory substrate: peptone (16 g), meat extract (11 g), urea (3 g), NaCl (0.7 g), CaCl2.2H2O (0.4 g), MgSO4.7H2O (0.2 g) and K2HPO4 (2.8 g) dissolved in 1L of water with the aid of ultrasonication.
- The pH of the synthetic sewage stock was 7.3 in the range-finding test. The pH of the synthetic sewage stock used to feed the activated sludge was 7.3 in the definitive test and 7.4 in the additional experiment.
- The pH was measured using a Hach HQ160 Flexi handheld meter.

PREPARATION OF INOCULUM
- 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 day of collection, the activated sludge (10 L) was fed synthetic sewage (500 mL).
- The pH of the sample was 7.3 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
- Filtration was performed using a Buchner funnel which was then rinsed three times with 10 mL of deionised reverse osmosis water and filtration was 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.
- The process of drying and weighing was repeated until a constant weight was obtained.
- 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
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
20 °C
pH:
Test item preparations after stirring and prior to addition of inoculum in the range-finding test were pH 7.0 to 7.4 after adjustment (see Table 1, attached)
Test item preparations after stirring and prior to addition of inoculum in the definitive test were pH 7.0 to 8.0 after adjustment (see Table 6, attached)
Test item preparations after stirring and prior to addition of inoculum in the additional test were pH 7.0 to 7.7 after adjustment (see Table 11, attached)
Dissolved oxygen:
Dissolved oxygen concentration of the test item preparations after 30 minutes contact time in the range-finding test were 71 to 92 % of the dissolved oxygen saturation level of 8.9 mg O2/L (see Table 2, attached).
Dissolved oxygen concentration of the test item preparations after 30 minutes contact time in the definitive test were 69 to 80 % of the dissolved oxygen saturation level of 8.9 mg O2/L (see Table 7, attached).
Dissolved oxygen concentration of the test preparations after 30 minutes contact time in the additional test were 65 to 74 % of the dissolved oxygen saturation level of 8.9 mg O2/L (see Table 12, attached).
Salinity:
Not applicable
Nominal and measured concentrations:
10, 100 and 1000 mg/L nominal concentration in range-finding test
56, 100 and 180 mg/L nominal concentration in definitive test
320 mg/L nominal concentration in additional experiment
Details on test conditions:
TEST WATER
- Deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC) was used for the range-finding, definitive tests and additional expirement.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol)
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
180 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Details on results:
RANGE-FINDING TEST
- The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2 (attached).
- Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in Table 3 (attached).
- The pH values of the test preparations at the start and end of the exposure period are given in Table 4 (attached).
- Observations made on the test preparations throughout the range-finding test are given in Table 5 (attached).
- 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.
- No statistically significant toxic effects were shown at test concentrations of 10 and 100 mg/L, however, statistically significant toxic effects were shown at 1000 mg/L.
- It was considered necessary to perform a definitive test in order to obtain a NOEC for the test item.
- Based on this information, test concentrations of 56, 100 and 180 mg/L were selected for the definitive test.

DEFINITIVE TEST
- The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 12 (attached).
- Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 13 hours contact time are given in Table 3 (attached).
- The pH values of the test preparations at the start and end of the exposure period are given in Table 14 (attached).
- Observations made on the test preparations throughout the range-finding test are given in Table 15 (attached).
- No statistically significant toxic effects were shown at test concentrations of 56, 100 and 180 mg/L.
- Therefore, an additional test was performed where statistically significant toxic effects (P<0.005) were shown at concentration of 320 mg/L.
- Based on these results, NOEC after 3 hours exposure was 180 mg/L.

VALIDATION CRITERIA FOR THE DEFINITIVE TEST
- The coefficient of variation of oxygen uptake in the control vessels was 9 % and the specific respiration rate of the controls was 26.39 mg oxygen per gram dry weight of sludge per hour.
- Validation criteria were satisfied.

- 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.
- 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.
Results with reference substance (positive control):
INHIBITION OF RESPIRATION RATE BY 3,5-DICHLOROPHENOL
- EC10 (3 h) 2.0 mg/L
- EC20 (3 h) 2.8 mg/L
- EC50 (3 h) 8.1 mg/L (95 % confidence limits 6.4-10 mg/L)
- EC80 (3 h) 23 mg/L
- The validation criterion for the reference item EC50 was also satisfied.
Reported statistics and error estimates:
ECx AND NOEC
- 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.
- Calculation of 95 % confidence limits for the reference item EC50 value was performed using the method of Litchfield and Wilcoxon (1949).
- 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)
Validity criteria fulfilled:
yes
Conclusions:
The Observed No Effect Concentration (NOEC) after 3 hours exposure was 180 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2010) No 209 “Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

METHODS

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 50, 100 and 180 mg/L (5 replicates) for a period of 3 hours at a temperature of between 21 to 22 °C with the addition of a synthetic sewage as a respiratory substrate.

An additional experiment was performed after the definitive test whereby activated sewage sludge was exposed to an aqueous dispersion of 320 mg/L (5 replicates) for a period of 3 hours at a temperature of between 19 to 20 °C with the addition of a synthetic sewage as a respiratory substrate in order to determine the No Observed Effect Concentration (NOEC).

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

RESULTS

The NO Observed Effect Concentration (NOEC) after 3 hours exposure was 180 mg/L.

The reference item gave a 3-hour EC50 value of 8.1 mg/L (95% confidence limits 6.4 to 10 mg/L).

Description of key information

The Observed No Effect Concentration (NOEC) after 3 hours exposure was 180 mg/L. (OECD 209).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
180 mg/L

Additional information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2010) No 209 “Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

METHODS

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 50, 100 and 180 mg/L (5 replicates) for a period of 3 hours at a temperature of between 21 to 22 °C with the addition of a synthetic sewage as a respiratory substrate.

An additional experiment was performed after the definitive test whereby activated sewage sludge was exposed to an aqueous dispersion of 320 mg/L (5 replicates) for a period of 3 hours at a temperature of between 19 to 20 °C with the addition of a synthetic sewage as a respiratory substrate in order to determine the No Observed Effect Concentration (NOEC).

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

RESULTS

The NO Observed Effect Concentration (NOEC) after 3 hours exposure was 180 mg/L.

The reference item gave a 3-hour EC50 value of 8.1 mg/L (95% confidence limits 6.4 to 10 mg/L).