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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 21-10-2014 Experimental completion date: 22-10-2014
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 (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch/Lot no. 1205442102
Appearance: Clear liquid
Solubility: Miscible in water (78-302 mg/L)
Volatile: No (vapour pressure 100 Pa, 35°C)
Stability: Unknown under test conditions
Storage: -20°C in the dark
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The tested nominal concentrations of the reference substance were 1.25, 2.5, 5, 10, 20 and 40 mg/L and 17.5, 34.5, 70.0, 138.0, 278.0, 552.0 and 1102.0 mg/L for the test substance.

The test was performed in 300 mL or 1 L Erlenmeyer flasks with total working volumes of 50 and 200 mL, respectively.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (21-10-2014) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The suspended solids (dry weight) content of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The activated sludge was aerated and fed
overnight with OECD synthetic sewage concentrate at 50 mL/L prior to use on the day following collection. The suspended solids concentration of the activated sludge in the incubation vessels was 1.5 g dry weight/L.
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Test temperature:
20°C
pH:
pH 6.9-7.0
Nominal and measured concentrations:
nominal concentrations of the reference substance were 1.25, 2.5, 5, 10, 20 and 40 mg/L and 17.5, 34.5, 70.0, 138.0, 278.0, 552.0 and 1102.0 mg/L for the test substance
Details on test conditions:
Test conditions
The test was performed in 300 mL or 1 L Erlenmeyer flasks with total working volumes of 50 and 200 mL, respectively. The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test compound and synthetic sewage.

Synthetic sewage feed and stocks
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water: 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. In the incubation vessels 1.6 or 6.4 mL of the synthetic sewage feed is added in a total volume of 50 and 200 mL, respectively. Tert-pentyl peroxypivalate was weighed directly in the incubation vessels. 3,5-Dichlorophenol was added to the incubation vessels using a stock solution of 1.0 g/L. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1003 g of 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Finally the pH was corrected with 1M H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.

Test procedures
The activated sludge respiration inhibition test was performed according to the study plan. The study plan was developed from an OECD Test Guideline (OECD 209, 2010). In this test the inhibition of oxygen uptake by micro-organisms oxidizing organic carbon was not separately expressed from the respiration by micro-organisms oxidizing ammonium. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. Some aspects of the test are listed below:
• Prior to use the activated sludge was homogenized with a syringe.
• The activated sludge with test substances was incubated in a shaking bath (100 rpm) for 3 hours.
• The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two. The tested nominal concentrations of the reference substance were 1.25, 2.5, 5, 10, 20 and 40 mg/L and 17.5, 34.5, 70.0, 138.0, 278.0, 552.0 and 1102.0 mg/L for the test substance.
• An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical breakdown of the test substance is expected.
• The test substance was pipetted into the incubation vessels and the added weight was directly measured on an analytical mass balance. The weight of the test substance volumes pipetted in the 1 L incubation vessels could not be directly determined. The average weight of four measurements of the same pipetted volume, determined in a 100 ml beaker glass, was used.
• Since the test substance was weighed directly into the incubation vessel and was considered unlikely to be air-stripped during incubation, no specific analyses were performed.

Determination of respiration rates, pH, dry weight and temperature
The respiration rates of the activated sludge were measured in a Biological Oxygen Monitor YSI 5300 (BOM). The BOM consisted of a thermostated vessel with a magnetic stirrer and an oxygen electrode. The electrode to measure the oxygen depletion tightly closed the vessel. The volume of the vessel was 10 mL and the temperature in the vessel was 20°C. The pH was measured using an EUTECH cyberscan pH11 pH meter. The temperature was measured with a Keithley 871A digital thermometer. The dry weight of the inoculum was determined by filtering 50 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter and retained sludge solids were dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. The concentration of suspended solids (dry weight) was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume.

Calculation of test results
The respiration rate was calculated from the linear part of the respiration curve as mg O2/L/min. Specific respiration rates (activity) in mg O2/g/min were calculated by dividing respiration rates by the dry weight sludge solids concentration in the incubation vessels.
In order to calculate the inhibitory effect of test substance at a particular concentration, the respiration rate was expressed as a percentage of the mean of two controls of the respiration rate:
(1 – 2Rs / (Rc1 + Rc2)) x 100 = percentage inhibition
Rs = oxygen-consumption rate at tested concentration of test substance.
Rc = oxygen-consumption rate of controls.
The EC values were computed from the best-fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the nominal concentration of the substance. All computations were performed with the program TOXCALCTM (Tidepool Scientific Software 1994-1999).
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
296 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
1 322 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The inhibitory effect of tert-pentyl peroxypivalate at a particular nominal concentration is expressed as a percentage of the two controls. From these results EC values were calculated. The EC50 of tert-pentyl peroxypivalate for activated sludge after 3 hours contact time is 296 mg/L with 95% confidence limits of 78 and 386771 mg/L. The EC10, EC20 and EC80 are 30, 66 and 1322 mg/L, respectively.
Results with reference substance (positive control):
The validity of the test is demonstrated by three validity criteria which were met according the guideline. First, the average oxygen uptake rate for the control replicates was 20.2 mg O2/g dry weight/hour, which is just meeting the criterion of >20 mg O2/g dry weight/hour. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and as a consequence has a low oxygen uptake rate. Operating at low organic sludge loads is nowadays very common. A lower oxygen
uptake rate does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate. A lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 4%, which shows a high precision and fulfills the maximum prescribed variation of <30%. Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 13 mg/L, which is within the prescribed range of 2 to 25 mg/L.

Test conditions

The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.3. Temperature varied from 20.1 to 20.9°C. These conditions allow respiration of the activated sludge used.

Study plan

There were no deviations from the study plan. There was one amendment to the study plan. The test substance was dosed by pipetting rounded volumes to the incubation vessels therefore the nominal test

substance concentrations tested were: 17.5, 34.5, 70.0, 138.0, 278.0, 552.0 and 1102.0 mg/L instead of 20, 40, 80, 160, 320, 640 and 1280 mg/L.

Table I: Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of tert-pentyl peroxypivalate after 3 hours contact time

Weighed test substance (mg)

Concentration (mg/L)

Activity

(mg O2/g/min)

Inhibition (%)

pH at start test

pH at end test

Temperature at end test (°C)

-

Control

0.3286

-

7.0

7.0

20.4

-

Control

0.3375

-

7.0

7.1

20.9

-

Control*

0.3381

-

6.9

7.0

20.5

-

Control*

0.3524

-

7.0

7.1

20.3

3.5# (0.00)

17.5

0.3238

3

7.0

7.1

20.3

6.9# (0.05)

34.5

0.2778

17

7.0

7.1

20.5

3.5

70.0

0.2571

23

6.9

7.2

20.1

6.9

138.0

0.2296

31

6.9

7.1

20.3

13.9

278.0

0.1704

49

6.9

7.2

20.7

27.6

552.0*

0.1222

65

7.0

7.3

20.4

55.1

1102.0*

0.0857

75

7.0

7.2

20.4

* = corresponding control and test substance concentration

#= Test substance pipetted in 1 L incubation vessels, average weighed and between brackets the standard deviation of four measurements of a pipetted volume of the test substance.

Table II: Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of 3,5-dichlorophenol after 3 hours contact time.

Concentration (mg/L)

Activity

(mg O2/g/min)

Inhibition (%)

pH at start test

pH at end test

Temperature at end test (°C)

Control

0.3167

-

7.0

7.1

20.3

Control

0.3286

-

7.0

7.0

20.4

Control*

0.3381

-

6.9

7.0

20.5

Control*

0.3524

-

7.0

7.1

20.3

1.25*

0.3083

11

7.0

7.0

20.3

2.5

0.2952

8

7.0

7.1

20.5

5.0

0.2875

11

7.1

7.0

20.2

10.0

0.2630

18

7.1

7.0

20.1

20.0

0.0815

75

7.1

7.0

20.4

40.1

0.0407

87

7.1

7.0

20.5

Concentration stock solution 3,5-dichlorophenol = 1000 mg/L

pH stock solution 3,5-dichlorophenol = 7.7

* = corresponding control and reference substance concentration

Validity criteria fulfilled:
yes
Conclusions:
The inhibitory effect of tert-pentyl peroxypivalate at a particular nominal concentration is expressed as a percentage of the two controls. From these results EC values were calculated. The EC50 of tert-pentyl peroxypivalate for activated sludge after 3 hours contact time is 296 mg/L with 95% confidence limits of 78 and 386771 mg/L. The EC10, EC20 and EC80 are 30, 66 and 1322 mg/L, respectively.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge

was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The test is valid as shown by the EC50 of the reference substance, 3,5-dichlorophenol (13 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (4%). The average

oxygen uptake rate for the control replicates is 20.2 mg O2/g dry weight/hour this is just above the prescribed rate (>20 mg O2/g dry weight /hour). The toxicity of tert-pentyl peroxypivalate to activated

sludge was determined at a contact time of 3 hours, using various concentrations of the test substance.

The inhibitory effect of tert-pentyl peroxypivalate at a particular nominal concentration is expressed as a percentage of the two controls. The EC50 of tert-pentyl peroxypivalate for activated sludge after 3 hours contact time is 296 mg/L with 95% confidence limits of 78 and 386771 mg/L. The EC10, EC20 and EC80 are 30, 66 and 1322 mg/L, respectively.

Description of key information

Toxicity to microorganisms was determined in an activated sludge respiration inhibition test was performed according to OECD 209 under GLP.

The EC50 of tert-amyl peroxypivalate for activated sludge after 3 hours contact time is 296 mg/L. The EC10, EC20 and EC80 are 30, 66 and 1322 mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:
296 mg/L
EC10 or NOEC for microorganisms:
30 mg/L

Additional information