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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: 04-04-2017 Experimental completion date: 05-04-2017
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))
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/lot No.: 0708501022
Purity: 99.8 %
Appearance: Red powder
Solubility: Not soluble in water (reacts with water under alkaline conditions, stable at neutral pH)
Volatile: Expected non-volatile (vapour pressure < 1.3 Pa)
The concentrations cited in the summary refer to the as-received sample of dichlorobis(r1-cylcopentadienyl)titanium.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Dichlorobis(η-cylcopentadienyl)titanium was weighed directly in the incubation vessels. 3,5-Dichlorophenol was added to the incubation vessels using a 1.0 g/L stock solution.
The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1008 g of 3,5-dichlorophenol (99.7%) in approximately 40 mL of deionized water with the addition of a
few drops of 2 M NaOH. Finally the pH was corrected with 2M H2SO to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (04-04-2017) 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 dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20°C
pH:
ca. pH 7
Conductivity:
>5 µS/cm
Nominal and measured concentrations:
Concentrations: 23, 42, 81, 162, 324 mg/L of test substance
Details on test conditions:
Test conditions
The test was performed in 300 mL Erlenmeyer flasks with a total working volume of 50mL. 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.

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, 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/min) for 3 hours.
- The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two.
- An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen clue to chemical breakdown of the test substance is expected.
- 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 (BOM) YSI 5300. 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 Testo 925 digital thermometer. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. 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. Divided by the activated sludge dry weight concentration in the incubation vessels a respiration rate (activity) in mg O2/g/min was calculated.
In order to calculate the inhibitory effect of a 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 (linear maximum likelihood regression) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance. All computations were performed with the program ToxRatPro version 2.1.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
712 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The inhibitory effect of dichlorobis (η-cylcopentadieny|)titanium at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of dichlorobis(η-cylcopentadienyl)titanium for activated sludge after 3 hours contact time is 712 mg/L with 95% confidence limits of 322 and 5631 mg/L. The EC10 and EC80 values could not be determined and the EC20 values was 23 mg/L. Dichlorobis(η-cylcopentadienyl)titanium is therefore considered not harmful to activated sludge.

Test conditions:

The pH of the reaction mixtures after the incubation period ranged from 6.4 to 7.1. Temperature varied from 19.9 to 20.4 °C. These conditions allow respiration of the activated sludge used.

Validity of the test

The validity of the test is demonstrated although only two out of three validity criteria were met according the guideline. First, the prescribed oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements was not met. 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. Nonetheless the average oxygen uptake rate for the control O2/g rate. This 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. The 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 7%, which shows a high precision and fulfils the maximum prescribed variation of <30%. Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 10 mg/L, which is within the prescribed range of 2 to 25 mg/L.

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

0.2667

-

6.8

7.0

20.3

Control

0.2524

-

6.8

7.1

19.9

2.5

0.2190

16

6.8

7.1

20.1

5.0

0.2000

23

6.8

7.1

20.1

10.1

0.1278

51

6.8

7.0

20.1

20.1

0.0667

74

6.8

7.1

20.2

40.2

0.0286

89

6.9

7.1

20.00

Concentration stock solution 3,5-dichlorophenol = 1005 mg/L

pH stock solution 3,5-dichlorophenol = 7.0

pH synthetic sewage = 7.1

Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of dichlorobis(ɳ-cylcopentadienyl)titanium 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

0.2524

-

6.8

7.1

19.9

-

Control

0.2889

-

6.9

7.0

20.2

-

Control*

0.2583

-

6.8

7.1

20.3

-

Control*

0.2333

-

6.8

7.0

20.3

1.15

23

0.2083

23

6.8

7.1

19.9

2.11

42

0.2278

16

6.9

7.1

20.4

4.04

81

0.1611

40

6.9

7.0

20.0

8.11

162

0.1833

32

6.9

7.0

20.2

16.19

324

0.1611

40

6.9

6.9

20.2

32.06

641*

0.1333

46

6.8

6.4

20.4

64.06

1281*

0.1000

59

6.8

6.5

20.2

* = corresponding control and reference concentration

Validity criteria fulfilled:
yes
Conclusions:
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 compound 3,5-dichlorophenol (10 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (7%). The average oxygen uptake rate for the control replicates is lower (16 mg O2/g dry weight/hour) than the prescribed rate (>20 mg O2/g dry weight/hour). This 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. The lower average oxygen uptake rate does therefore does 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.
The toxicity of dichlorobis(ɳ-cylcopentadienyl)titanium to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of dichlorobis(ɳ-cyclopentadienyl)titanium at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of dichlorobis (ɳ-cylcopentadienyl)titanium for 3 hours contact time is 712 mg/L with 95% confidence limits of 322 and 5631 mg/L. The EC10 and EC80 values could not be determined and the EC20 values was 23 mg/L. Dichlorobis(ɳ-cylcopentadienyl) is therefore considered not harmful to activated sludge.
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 compound 3,5-dichlorophenol (10 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (7%). The average oxygen uptake rate for the control replicates is lower (16 mg O2/g dry weight/hour) than the prescribed rate (>20 mg O2/g dry weight/hour). This 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. The lower average oxygen uptake rate does therefore does 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.

The toxicity of dichlorobis(ɳ-cylcopentadienyl)titanium to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of dichlorobis(ɳ-cyclopentadienyl)titanium at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of dichlorobis (ɳ-cylcopentadienyl)titanium for 3 hours contact time is 712 mg/L with 95% confidence limits of 322 and 5631 mg/L. The EC10 and EC80 values could not be determined and the EC20 values was 23 mg/L. Dichlorobis(ɳ-cylcopentadienyl) is therefore considered not harmful to activated sludge.

Description of key information

The EC50of dichlorobis (ɳ-cylcopentadienyl)titanium for 3 hours contact time is 712 mg/L with 95% confidence limits of 322 and 5631 mg/L. The EC10and EC80values could not be determined and the EC20values was 23 mg/L. Dichlorobis(ɳ-cylcopentadienyl) is therefore considered not harmful to activated sludge.

Key value for chemical safety assessment

EC50 for microorganisms:
712 mg/L

Additional information