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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-14 - 2016-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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)
Remarks:
Department of Health of the Government of the United Kindom
Analytical monitoring:
no
Details on sampling:
After a contact time of 3 hours, an aliquot (250 mL) was transferred into a BOD bottle containing a magnetic stir bar. The rate of respiration was measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace. 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.
Vehicle:
no
Details on test solutions:
Preparation of Test Item:
Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 22 °C, in order to maximize the dissolved test item 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). The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 and 8.0 if necessary

Preparation of the Reference Item:
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 5.9 and was adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Preparation of Mineral Oil Control:
Additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations; 5.3, 53 and 530 mg/L. The test item was dispersed directly in water. Nominal amounts of test item (2.65, 26.5 and 265 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 22 °C, in order to maximize the dissolved test item 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). The pH of the test item dispersions was measured after stirring using 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: Severn Trent Water Plc sewage treatment plant, Loughborough, Leicestershire, UK.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample 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 using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.

-Test Water
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

-Synthetic Sewage
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock was 7.2. The pH value was measured using a Hach HQ40d Flexi handheld meter.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period reported.
Hardness:
Not reported
Test temperature:
20 - 20 °C
pH:
7.4 - 7.8
Dissolved oxygen:
After a contact time of 3 hours, an aliquot (250 mL) was transferred into a BOD bottle containing a magnetic stir bar. The rate of respiration was measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace. 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.
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
10, 100, 1000 mg/L
Details on test conditions:
Preparation of Test System
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 with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter 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. The test item vessels were prepared as described in Section 3.3.1.1 followed by the mineral oil controls as described in Section 3.3.1.3. Two further control vessels were prepared. Finally three additional mineral oil control vessels were then prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 and 21 ºC.

Activated sewage sludge was exposed to an aqueous dispersion of the test item 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 between 20 and 21 °C with the addition of a synthetic sewage as a respiratory substrate.
The test item was a complex mixture containing 53% mineral oil. Additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations; 5.3, 53 and 530 mg/L.
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.

Calculation of the Oxygen Uptake Rates
The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:
R = [(Q1-Q2) / Δt] * 60

where:
Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt = the time interval between the beginning and end of the selected section of the linear phase (min).

The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:
RS = R / SS

where:
SS = the concentration of suspended solids in the test mixture (g dry weight/L).

Calculation of Percentage of Inhibition
The percentage inhibition was calculated according to the following equation:
% inhibition = [1 – (R/Rbc)] x 100

where:
Rbc = the mean respiration rate of the blank controls

ECX and NOEC
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. 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (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 test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups to see where the NOEC lies. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
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. (total fraction)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Blank controls oxygen uptake rate:
The initial O2 reading of the four controls was between 5.7 and 6.4 mg O2/L. After a measurement period of 6 to 7 minutes, final O2 readings indicated a decrease of oxygen to 1.8 - 2.1 mg O2 / L. This equates a mean O2 consumption rates of approximately 37.7 mg O2 / (L * hour).
Results with reference substance (positive control):
As reference substance, 3,5-dichlorophenol was used at three concentrations: 3.2, 10, 32 mg/L. Thereby, the O2 consumption rates of approximately 29.3, 18.6 and 4.8 mg O2/ (L * hour)) were obtained for the 3,5-dichlorophenol concentration of 3.2, 10 and 32 mg/L, respectively. Also, controls were measured that were inoculated with mineral oil at three concentrations: 5.3, 53 and 530 mg/L. However, the nominal concentration of the mineral oil had no effect on the O2 consumption rate which was approximately 36,6 mg O2 / (L*hour).
Reported statistics and error estimates:
The coefficient of variation of oxygen uptake in the control vessels was 2.74% and the specific respiration rate of the controls was 25.12 mg oxygen per gram dry weight of sludge per hour.

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test.

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 5.9 6 2.1 38.00 -
R2 6.1 7 1.8 36.86 -
R3 6.4 7 2.1 36.86 -
R4 5.7 6 1.8 39.00 -
Test Item 10 5.1 5 2.0 37.20 1
100 4.4 4 1.8 39.00 [4]
1000R1 4.1 3 2.1 40.00 [6]
1000R2 2.7 1 2.0 42.00 [11]
1000R3 5.2 5 2.2 36.00 4
3,5-dichloro- phenol 3.2 6.4 9 2.0 29.33 22
10 7.5 10 4.4 18.60 51
32 8.3 10 7.5 4.80 87
Mineral Oil Control 5.3 6.3 7 2.0 36.86 2
53 5.3 6 1.7 36.00 4
530 5.7 6 2.0 37.00 2

[Increase in respiration rate as compared to controls]

R1 – R4 = Replicates 1 to 4

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.

No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

No statistically significant toxic effects were shown at all of the mineral oil control test concentrations employed.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Inhibition of Respiration Rate

The following results were derived:

Test item Mineral Oil Control 3,5-dichlorophenol
ECx (3 Hours) (mg/L) 95%Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
>1000 - >530 - 2.2 -
>1000 - >530 - 3.1 -
>1000 - >530 - 9.0 7.1 - 11
>1000 - >530 - 26 -
1000 - 530 - - -
Validity criteria fulfilled:
yes
Remarks:
Scientifically validity criteria were met.
Conclusions:
The coefficient of variation of oxygen uptake in the control vessels was 2.74% and the specific respiration rate of the controls was 25.12 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
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

A GLP 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 the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)". Activated sewage sludge was exposed to an aqueous dispersion of the test item 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 between 20 and 21 °C. Synthetic sewage was addad as a respiratory substrate. 3,5-dichlorophenol was used as reference item (concentrations: 3.2, 10, 32 mg/L) and additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations: 5.3, 53 and 530 mg/L. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item. Observations were made on the test preparations throughout the test period of 3 hours. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge. As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into a BOD bottle and 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 a magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace. The mean O2 consumption rates of the four control samples was approximately 37.7 mg O2 / (L * hour). The reference substance 3,5-dichlorophenol exhibited O2 consumption rates of 29.3, 18.6 and 4.8 mg O2 / (L * hour)) for its concentration of 3.2, 10 and 32 mg/L, respectively. The nominal concentration of the mineral oil had no effect on the O2 consumption rate which was approximately 36.6 mg O2 / (L*hour). The coefficient of variation of oxygen uptake in the control vessels was 2.74% and the specific respiration rate of the controls was 25.12 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. The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The reference item gave a 3-Hour EC50 value of 9.0 mg/L, 95% confidence limits 7.1 to 11 mg/L.

Description of key information

Toxicity to Activated Sludge in a Respiration Inhibition Test, OECD 209, static: EC50 (3h) > 1000 mg/L nominal, NOEC (3h) 1000 mg/L nominal

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

A GLP key study (Bayliss, 2016b) 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 the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)". Activated sewage sludge was exposed to an aqueous dispersion of the test item 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 between 20 and 21 °C. Synthetic sewage was added as a respiratory substrate. 3,5-dichlorophenol was used as reference item (concentrations: 3.2, 10, 32 mg/L) and additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations: 5.3, 53 and 530 mg/L. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item. Observations were made on the test preparations throughout the test period of 3 hours. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge. As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into a BOD bottle and 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 a magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace. The mean O2 consumption rates of the four control samples was approximately 37.7 mg O2 / (L * hour). The reference substance 3,5-dichlorophenol exhibited O2 consumption rates of 29.3, 18.6 and 4.8 mg O2 / (L * hour)) for its concentration of 3.2, 10 and 32 mg/L, respectively. The nominal concentration of the mineral oil had no effect on the O2 consumption rate which was approximately 36.6 mg O2 / (L*hour). The coefficient of variation of oxygen uptake in the control vessels was 2.74% and the specific respiration rate of the controls was 25.12 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. The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The reference item gave a 3-Hour EC50 value of 9.0 mg/L, 95% confidence limits 7.1 to 11 mg/L.