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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
and EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sapringly Soluble Chemicals)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Not conducted.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test concentrations (10, 31, 100, 313 and 1000 mg/L) were directly weighed into the designated test flasks to reach the planned nominal concentrations. The nominal test item concentrations were prepared by mechanical dispersion (using shortly (5 min) ultrasonic bath). These test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.

- Eluate: Deionised water
- Differential loading: See table 1 below.
- Controls: Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 10, 31, 100, 313 and 1000 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary
Conditioning: The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice.

An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.43. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not measured.
Test temperature:
The temperature was measured in the climate chamber with a min/max thermometer during the incubation period.
19.8 – 20.9 deg C (during the incubation) and
20.3 – 21.1 deg C (during oxygen measurement)
pH:
The pH was determined at the start and at the end of the incubation period in all treatments. Values are detailed in the results section in Table 2 below.
Dissolved oxygen:
Dissolved oxygen was determined at the start and at the end of the incubation period in all treatments. Values are detailed in the results section in Table 2 below.
Salinity:
Not applicable.
Nominal and measured concentrations:
The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item/L were not tested. Composition of Test Media specifications are detailed in Table 1, below.
Details on test conditions:
Test conditions

Surrounding type:Climate chamber (during the incubation) and controlled environment room (during the formulation and oxygen measuring)
Temperature: 19.8 – 20.9 deg C (during the incubation) and
20.3 – 21.1 deg C (during oxygen measurement)
Aeration: With compressed air (approximately 1 litre per minute)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

Test units

Type and size: Erlenmeyer bottles of approximately 350 mL volume, and BOD bottles with special neck of 300 mL volume.
Identification: Each test flask was uniquely identified with at least study code, treatment and replicate codes (in case of controls).

Equipment used

Normal laboratory equipments were used in the study,
Self stirring O2 electrode,
Oxygen meter,
Thermometer,
pH meter,
Balance,
Centrifuge,
Moisture analyzer,
Aeration system,
Orbital shaker

Preparations of the test flasks

One test solution with a final volume of 330 mL was tested per treatment in a glass flask. 10.56 mL synthetic sewage and an adequate amount of the test item or an adequate volume of the stock solution of the reference item was filled up with deionised water to 198 mL before the start of the test. At the start of the test 132 mL activated sludge inoculum with a sludge concentration of 4 g/L (dry weight) was added, first to first control (C1), then in time intervals of 15 minutes (an arbitrary but convenient interval) to the test solutions of the reference item and the test item and finally to a second control (C2). Time interval between the last test solution flask and the second control was more than 15 minutes.

Synthetic Sewage Feed (ratio of composition of culture media referring to 1000 mL)

Peptone 16.0 g
Meat extract 11.0 g
Urea 3.0 g
NaCl 0.7 g
CaCl2 x 2H2O 0.4 g
MgSO4 x 7H2O 0.2g
K2HPO4 2.8 g
Deionised water add 1000.0 mL

Measurement of Respiration Rate

For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with a stirring O2 electrode and was recorded for about ten minutes. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

INHIBITION OF THE RESPIRATION RATE

For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with a stirring O2 electrode and was recorded for about ten minutes. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.


Measurement of pH, Dissolved Oxygen and Water Temperature

The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all treatments. The temperature was measured in the climate chamber with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all BOD bottles.

Reference Control (R1 – R3)

In parallel to the study with the test item, the reference item 3,5-Dichlorophenol was tested (the nominal test concentrations of 5, 16 and 32 mg/L) under otherwise identical test conditions.
A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209: 0.25 g of 3,5-Dichlorophenol was dissolved in 5 mL 1 mol/L NaOH and diluted to about 15 mL with deionised water. Excess of NaOH was neutralised with approximately 4 mL of 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to 0.5 litre with deionised water. The final pH was measured to be 7.61 and the final concentration amounted 500 mg/L.

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.
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.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Test item
In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 1.8 % and 7.0 % in the examined nominal test concentration range (10 - 1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested.
The influence of 4,4-diaminodiphenylether on the respiration rate of activated sludge in Table 2 and in Figure 1, below

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

VALIDITY CRITERIA

-The respiration rates of the two controls did not differ by more than 15 %
-The 3-hour EC50 of the reference item 3,5-Dichlorophenol for the used activated sludge batch was determined to be in the range of 5 to 30 mg/L.
-The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.0 mg O2/L.

Results with reference substance (positive control):
The following nominal concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 5, 16 and 32 mg/L. In comparison to the controls the respiration rate of the activated sludge was inhibited by 35.1 % at the lowest nominal concentration of 5 mg/L.

At the nominal concentrations of 16 and 32 mg/L, the respiration rate was inhibited by 54.4 % and 71.9 %, respectively. The results are summarised in Table 3 and in Figure 1-2.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 11.46 mg/L with 95 % confidence limits of 8.48 to 15.49 mg/L.

The influence of the reference item on the respiration rate of activated sludge in Table 3 and in Figure 1-2, detailed below.
Reported statistics and error estimates:
The inhibitory effect of the test item respectively of the reference item at a particular concentration on the respiration rate were expressed as percentage of the mean value of the respiration rates of the two controls according to:

[1-(2Rs / [Rc1+Rc2]= x 100 = % inhibition

where:

Rs = oxygen consumption rate at tested concentration of test item
Rc1 = oxygen consumption rate of control 1
Rc2 = oxygen consumption rate of control 2

The 3-hour EC20 of the test item and the 3-hour EC50 of the reference item with 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

The 3-hour EC20, EC50 and EC80 of the test item could not be calculated.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (7.0 %) at the concentration level of 313 and 1000 mg/l was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.530 (at 313 and 1000 mg/l) was in the historical control data range (0.516 - 0.074), and the deviation from the control was within +/- 15 %, can be considered as a biological variability of the test system.



The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (7.0 %) at the concentration level of 313 and 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.530 (at 313 and 1000 mg/L) was in the historical control data range (0.516 - 0.074), and the deviation from the control was within +/- 15 %, can be considered as a biological variability of the test system.

Table 2.         Influence of test item on oxygen consumption of activated sludge

 

Flask
No.

ID

Test group

Concentration
(mg/L)

Oxygen consumption
(mg O2/L/min)

Inhibition
(%)

pH-values

Oxygen concentration
(mg O2/L)

start *

end *

start *

end *

1

C1

Control

0.580

7.34

7.33

7.9

7.9

15

C2

Control

0.560

7.31

7.39

7.8

7.9

 

Mean

0.570

 

Deviation (%)

3.5

5

T1

Test item

10

0.560

1.8

7.28

7.21

7.7

7.8

6

T2

Test item

31

0.550

3.5

7.22

7.26

7.6

7.7

7

T3

Test item

100

0.540

5.3

7.24

7.42

7.5

7.5

8

T4

Test item

313

0.530

7.0

7.23

7.53

8.0

7.7

9

T5

Test item

1000

0.530

7.0

7.22

7.45

7.4

7.6

*     start and end of 3-hour aeration

ID:        Code of each group

 

Table 3.         Influence of reference item on oxygen consumption of activated sludge

 

Flask
No.

ID

Test group

Concentration
(mg/L)

Oxygen consumption
(mg O2/L/min)

Inhibition
(%)

pH-values

Oxygen concentration
(mg O2/L)

start *

end *

start *

end *

1

C1

Control

0.580

7.34

7.33

7.9

7.9

15

C2

Control

0.560

7.31

7.39

7.8

7.9

 

Mean

0.570

 

Deviation (%)

3.5

2

R1

Ref. item

5

0.370

35.1

7.70

7.88

7.9

7.0

3

R2

Ref. item

16

0.260

54.4

7.33

7.63

8.0

7.7

4

R3

Ref. item

32

0.160

71.9

7.24

7.53

7.9

7.2

*     start and end of 3-hour aeration

ID:        Code of each group

Validity criteria fulfilled:
yes
Remarks:
The respiration rates of the two controls did not differ by more than 15 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol for the used activated sludge batch was determined to be in the range of 5 to 30 mg/L.
Conclusions:
A laboratory test was carried out with 4,4-oxidianilline to evaluate the effect of the test item on microorganisms by measuring the respiration rate.
Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L.The substance did not inhibit the activated sludge used within the test.
Executive summary:

A laboratory test was carried out with 4,4-oxidianilline to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item/L were not tested.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 1.8 % and 7.0 % in the examined nominal test concentration range (10 - 1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested.

In parallel to the study with the test item, the reference item 3,5-Dichlorophenol was tested (the nominal test concentrations of 5, 16 and 32 mg/L) under otherwise identical test conditions.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 11.46 mg/L with 95 % confidence limits of 8.48 to 15.49 mg/L.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L.The substance did not inhibit the activated sludge used within the test.

Description of key information

A laboratory test was carried out with 4,4 -oxidianilline to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item/L were not tested.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 1.8 % and 7.0 % in the examined nominal test concentration range (10 - 1000 mg/L).

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L. The substance did not inhibit the activated sludge used within the test.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information