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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May 2015 to 06 October 2015
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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
RANGE FINDING TEST
- Nominal amounts of test item (5, 50 and 500 mg) were separately dispersed in approximately 200 mL of deionised reverse osmosis water in triplicate.
- Ultrasonication was applied for approximately 15 minutes followed by magnetic stirring for 24 hours at temperatures between 21 °C and 22 °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 if necessary.
- As it was not a requirement of the test guidelines, no analysis was conducted to determine the homogenicity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the compliance statement.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- Based on the results of the range-finding test, test concentrations were assigned to the definitive test as 100, 180 and 320 mg/L
- Nominal amounts of test item (50, 90 and 160 mg) were separately dispersed in approximately 200 mL of deionised reverse osmosis water (five replicates of each).
- 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 100, 180 and 320 mg/L (five replicates of each).
- 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 8.0 if necessary.
- The control group was maintained under identical conditions but not exposed to the test item.
- At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum was added in a 500 mL conical flask (first control).
- 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 per minute.
- Thereafter, at 15 minute intervals, the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added.
- Two additional control vessels were then prepared prior to the test item vessels being prepared.
- Finally, two further control vessels were prepared.
- The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 19 and 20 °C.

ADDITIONAL EXPERIMENT
- An additional experiment was performed at a concentration of 560 mg/L in order to confirm the No Observed Effect Concentration determined from the definitive test.
- A nominal amount of test item (280 mg) was dispersed in approximately 200 mL of deionised reverse osmosis water (five replicates).
- Ultrasonication was applied for approximately 15 minutes followed by magnetic stirring for 24 hours at approximately 20 °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 560 mg/L (five 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 8.0 if necessary.
- 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 21 August 2015 for the range-finding test, on 01 September 2015 for the definitive test and on 07 September 2015 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
- A synthetic sewage composed of 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) was prepared by dissolving in 1L of water with the aid of ultrasonication.
- In the range-finding test, the pH of the synthetic sewage stock was 7.1.
- In the definitive test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was 7.1 and the pH of the synthetic sewage stock used for the test was 7.2.
- In the additional experiment, the pH of the synthetic sewage stock used to feed the activated sewage sludge was 7.0 and the pH of the synthetic sewage stock used for the additional experiment was 7.1.
- The pH values were measured using a Hach HQ40d 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 the day of collection the activated sewage sludge (10 L) was fed synthetic sewage (500 mL).
- The pH of the sample on the day of the test was 7.9 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 that had been rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven.
- 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:
None
Hardness:
Not reported
Test temperature:
20 to 21 °C
pH:
- pH values of the test preparations in the definitive test were 7.7 to 7.8 (start) and 8.2 to 8.3 (end). Values relating to each replicate are reported in Table 9 (attached).
- pH values of the test preparations in the additional experiment were 7.2 to 7.3 (start) and 7.4 to 7.6 (end). Values relating to each replicate are reported in Table 14 (attached).
Dissolved oxygen:
- Dissolved oxygen concentrations of test item preparations after 30 minutes contact time in the definitive test were 62 to 80 % of dissolved oxygen saturation level of 8.9 mg O2/L (see Table 7, attached).
- Dissolved oxygen concentrations of test item preparations after 30 minutes contact time in the additional experiment were 67 to 90 % of dissolved oxygen saturation level of 8.9 mg O2/L (see Table 12, attached).
Salinity:
Not applicable
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal concentrations of 10, 100 and 1000 mg/L

DEFINITIVE TEST
- Nominal concentrations of 100, 180 and 320 mg/L

ADDITIONAL EXPERIMENT
- Nominal concentration of 560 mg/L

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 test, definitive test and additional experiment.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
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 % of the dissolved oxygen saturation lvel of 8.9 mg O2/L.
- No statistically significant toxic effects were shown at the test concentration of 10 and 100 mg/L. However, statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
- Based on these results, and after discussion with the sponsor, it was considered necessary to perform a definitive test in order to obtain a NOEC value for the test item.
- Test concentrations of 100, 180 and 320 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 7 (attached).
- Oxygen consumption rates and percentage inhibition values for the control, test and reference items after 3 hours contact time are given in Table 8 (attached).
- The pH values of the test preparations at the start and end of the exposure period are given in Table 9 (attached) and observations made on the test preparations throughout the definitive test are given in Table 10 (attached).
- Percentage inhibition is plotted against concentration for the reference item (3,5-dichlorophenol) in Figure 1 (attached).

ADDITIONAL 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 items after 3 hours contact time are given in Table 13 (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 additional test are given in Table 15 (attached).
- Percentage inhibition is plotted against concentration for the reference item (3,5-dichlorophenol) in Figure 2 (attached).

VALIDATION CRITERIA
- The coefficient of variation of oxygen uptake in the control vessels was 7.9% and the specific respiration rate of the controls was 22.25 mg oxygen per gram dry weight of sludge per hour.
- Validation criteria were satisfied.

INHIBITION OF RESPIRATION RATE
- No statistically significant toxic effects were shown at the test concentrations of 100, 180 and 320 mg/L. Therefore, an additional experiment was performed where statistically significant toxic effects (P < 0.05) were shown at the test concentration of 560 mg/L. Based on these results the no observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.
- In some instances, the intitial 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 concentration after 30 minutes contact time in all vessels were above 60 % 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 (3h) 2.2 mg/L
- EC20 (3h) 3.4 mg/L
- EC50 (3h) 13 mg/L (95 % confidence limits 9.7-17 mg/L)
- EC80 (3h) 49 mg/L
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 for the range-finding and definitive 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).
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge was assessed in accordance with OECD Guideline 209. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 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 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 100, 180 and 320 mg/L (5 replicates) for a period of 3 hours at temperatures of between 20 to 21 °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 560 mg/L (5 replicates) for a period of 3 hours at temperatures of between 20 to 21 °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 reference item, 3 -5 -dichlorophenol.

RESULTS

The No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 320 mg/L. The reference item gave a 3 -hour EC50 value of 13 mg/L, 95 % confidence limits of 9.7 to 17 mg/L.

CONCLUSION

The No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 320 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge was assessed in accordance with OECD Guideline 209.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
320 mg/L

Additional information

No additional data.