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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 to 02 December 2016
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:
TEST WATER
- The test water used for the range finding tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
- A mixed population of activated sewage sludge micro-organisms was obtained on 15 September 2016 for the initial range-finding test and on 2 December 2016 for the second range-finding test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
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:
Approximately 21 °C
pH:
7.7 to 7.9 (see Table 3, attached)
Dissolved oxygen:
1.9 to 6.4 mg O2/L (see Table 3, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentrations of 10, 100 and 1000 mg active ingredient(ai)/L
Details on test conditions:
SYNTHETIC SEWAGE
- Synthetic sewage was added to each test vessel to act as a respiratory substrate.
- The synthetic sewage contained 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 a final volume of 1 L water with the aid of ultrasonication.
- The pH of the synthetic sewage stock used in the initial range-finding test and range-finding test was 7.3 measured using a Hach HQ40d Flexi handheld meter.

INITIAL RANGE FINDING TEST – TEST ITEM PREPARATION
- In the initial 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 item was dissolved directly in water. However, the test item contained 41 % water, therefore a further range-finding test was performed to correct for the water content in the test item.

SECOND RANGE FINDING TEST – TEST ITEM PREPARATION

- In the second range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg ai/L. The test item was dissolved directly in water.
- A nominal amount of test item (4237 mg) was dissolved in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 liter to give a 2500 mg ai/L stock solution from which dilutions were made to give 250 and 25 mg ai/L stock solutions. An aliquot (200 mL) of the 25 mg ai/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg ai/L. Similarly, aliquots (200 mL) of the 250 mg ai/L and 2500 mg ai/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg ai/L. The 1000 mg ai/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.
- The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter (see Table 1, attached).
- The exposure conditions for each flask were as described below.
- As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This was an exception with regard to GLP and was reflected in the GLP compliance statement.
- The control group was maintained under identical conditions but not exposed to the test item.

RANGE-FINDING TEST – REFERENCE ITEM PREPARATION
- 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 INOCULUM
- The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection.
- The pH of the sample 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 deionized reverse osmosis water prior to drying in an oven) 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.

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 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.
- The test item vessels were prepared as described for the second range-finding test.
- Finally, two further control vessels were prepared.
- The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of approximately 21 ºC.

OBSERVATIONS
- Observations were made on the test preparations throughout the test period.
- Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.

MEASUREMENT OF pH
- 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.

OXYGEN CONCENTRATION
- Oxygen concentrations in all vessels were measured after 30 minutes contact time (see Table 2, attached).

MEASUREMENT OF RESPIRATION RATE
- 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.
- 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.

CALCULATION OF OXYGEN UPTAKE RATES
- The respiration rate, R, expressed in milligrams oxygen per litre per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph using the equation R = [(Q1 – Q2) / Δt] x 60 where Q1 = oxygen concentration at the beginning of the selected section of the linear phase (mg/L); Q2 = oxygen concentration at the end of the selected section of the linear phase (mg/L); Δt = 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 using the equation Rs = R / SS where SS = concentration of suspended solids in the test mixture (g dry weight/L).

CALCULATION OF PERCENTAGE INHIBITION
- Percentage inhibition was calculated using the equation % inhibition = [1 – (R/Rbc)] x 100 where Rbc = mean respiration rate of the blank controls.

VALIDATION CRITERIA
- Results of the study are considered valid if:
(i) The EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L for total respiration.
(ii) The specific respiration rate of the blank controls is not less than 20 mg oxygen per gram dry weight of sludge per hour for total respiration.
(iii) The coefficient of variation of oxygen uptake rate in control replicates is not more than 30% at the end of the test.

MAJOR COMPUTERISED SYSTEM
- Building management: Delta control system
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
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:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
INITIAL RANGE FINDING TEST
- In the initial range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L and no statistically significant toxic effects were shown at all of the test concentrations employed . However, the test item contained 41 % water, therefore a further range-finding test was performed to correct for the water content in the test item.

SECOND 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).
- 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 all 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 ai/L.
- Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 1, attached).
- 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 coefficient of variation of oxygen uptake in the control vessels was 0 % and the specific respiration rate of the controls was 28.00 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.
Results with reference substance (positive control):
- The EC10, EC20, EC50 and EC80 values calculated for the reference substance are shown in the table below.
Reported statistics and error estimates:
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.
- 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).

RESULTS WITH REFERENCE ITEM (3,5-DICHLOROPHENOL)

 

ECx (3h) mg/L

95 % confidence limits (mg/L)

EC10

1.8

-

EC20

2.6

-

EC50

8.1

6.3 to 10

EC80

25

-

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg ai/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L. The reference item gave a 3-Hour EC50 value of 8.1 mg/L, 95% confidence limits 6.3 to 10 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 the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

 

METHODS

In an initial range-finding test activated sewage sludge was exposed an aqueous solution 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 approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate. However, the test item supplied contained 41% water therefore a further range-finding test was performed where activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg active ingredient(ai)/L (3 replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at measured temperatures of approximately 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 item, 3,5-dichlorophenol.

 

RESULTS

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg ai/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L. The reference item gave a 3-Hour EC50 value of 8.1 mg/L, 95% confidence limits 6.3 to 10 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg ai/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L. The reference item gave a 3-Hour EC50 value of 8.1 mg/L, 95% confidence limits 6.3 to 10 mg/L (OECD 209).

Key value for chemical safety assessment

Additional information

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 the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

 

METHODS

In an initial range-finding test activated sewage sludge was exposed an aqueous solution 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 approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate. However, the test item supplied contained 41% water therefore a further range-finding test was performed where activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg active ingredient(ai)/L (3 replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at measured temperatures of approximately 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 item, 3,5-dichlorophenol.

 

RESULTS

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg ai/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L. The reference item gave a 3-Hour EC50 value of 8.1 mg/L, 95% confidence limits 6.3 to 10 mg/L.