Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, OECD & US EPA test guidance in compliance with GLP and reoprted with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F03-0318
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Before the start of the test defined amounts of the test item were dosed into the test flasks in a form of stock solution (2500 mg/L) by mechanical dispersion.
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.

Test solutions

Vehicle:
yes
Details on test solutions:
Just before the start of the test a concentrated stock solution (2500 mg/L) was prepared by dissolving test item in deionised water.
The test solutions used in the test were freshly prepared by dilution of the stock solution by mechanical dispersion at the beginning of the experiment, in the testing laboratory.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
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 7.41. The activated sludge was used directly after conditioning.

Study design

Test type:
not specified
Water media type:
brackish water
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period

Test conditions

Hardness:
No data
Test temperature:
19.2 – 20.4°C (during the incubation) and
19.4 – 20.5°C (during oxygen measurement)
pH:
7.45 - 7.60
Dissolved oxygen:
7.6 - 7.9 mg O2/l - start
7.1 - 7.5 mg O2/l finish
Salinity:
No data
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.
Details on test conditions:
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).

Test conditions
Surrounding type: Climate chamber (during the incubation) and controlled environment room (during the formulation and oxygen measuring)
Temperature: 19.2 – 20.4°C (during the incubation) and 19.4 – 20.5°C (during oxygen measurement)
Aeration: With compressed air (1 litre per minute)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

REFERENCE ITEM
Reference item: 3,5-Dichlorophenol
Supplier: SIGMA-ALDRICH
Batch No.: 15809KI
Expiry Date: 30 August 2012
Storage: In original container, at room temperature

CONTROLS
Untreated Control (C1 and C2)
Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.

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 7.45 and the final concentration amounted 500 mg/L.

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 stock solution 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 third reference item flask and the first test item solution flask was more than 15 minutes.

Synthetic Sewage Feed (ratio of composition of culture media referring to 1000 mL)
Peptone: 16.0g
Meat extract: 11.0g
Urea: 3.0g
NaCl: 0.7g
CaCl2 x 2H2O: 0.4g
MgSO4 x 7H2O: 0.2g
K2HPO4: 2.8g
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.

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 substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
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
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:
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.

Test item
In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited and a slight stimulation of respiration rates (-4.9; -2.9; -2.9 and -1.0%) were observed in the test concentration range of 10-313 mg Reactive Red F03-0318/L. The respiration rate was inhibited 2.9% at the highest test concentration of 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.
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 28.2% 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 59.2% and 78.6%, respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 11.32 mg/L with 95% confidence limits of 9.15 to 14.00 mg/L.
Reported statistics and error estimates:
The respiration rates of the two controls did not differ by more than 15% (5.85%).
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 (11.32 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 7.1 mg O2/L.

Any other information on results incl. tables

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

0.500

7.54

7.60

7.6

7.1

15

C2

Control

0.530

7.48

7.43

7.8

7.5

 

Mean

0.515

 

Deviation (%)

5.85

10

T1

Test item

10

0.540

-4.9

7.52

7.50

7.7

7.4

11

T2

Test item

31

0.530

-2.9

7.56

7.52

7.9

7.1

12

T3

Test item

100

0.530

-2.9

7.51

7.53

7.8

7.3

13

T4

Test item

313

0.520

-1.0

7.47

7.51

7.9

7.3

14

T5

Test item

1000

0.500

2.9

7.45

7.56

7.8

7.5

* start and end of 3-hour aeration

ID: Code of each group

 

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

0.500

7.54

7.60

7.6

7.1

15

C2

Control

0.530

7.48

7.43

7.8

7.5

 

Mean

0.515

 

Deviation (%)

5.85

2

R1

Ref. item

5

0.370

28.2

7.50

7.60

7.2

7.2

3

R2

Ref. item

16

0.210

59.2

7.52

7.62

7.6

7.7

4

R3

Ref. item

32

0.110

78.6

7.58

7.60

7.8

8.3

* start and end of 3-hour aeration

ID: Code of each group

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
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.
Executive summary:

A laboratory test was carried out with Reactive Red F03-0318 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

 

The study was performed in accordance with the study plan agreed upon by Sponsor, the OECD Guidelines for Testing of Chemicals (No. 209, 04 April 1984), the Council Regulation (EC) No 440/2008, Part C, C.11, the Product Properties Test Guideline OPPTS 850.6800 of the United States Environment Protection Agency (EPA) and the Principles of Good Laboratory Practice (GLP) and reoprted with a valid GLP certificate.

 

The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50.

 

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited and a slight stimulation of respiration rates (-4.9; -2.9; -2.9 and -1.0%) were observed in the test concentration range of 10-313 mg Reactive Red F03-0318/L. The respiration rate was inhibited 2.9% at the highest test concentration of1000 mg/L, which wasconsidered to reflect the biological variability of the test system and not to be a toxic effect on bacteria. 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 11.32 mg/L with 95% confidence limits of 9.15 to 14.00 mg/L.

 

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.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were not inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (2.9%) at the concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.500 (at 1000 mg/L) was below in the historical control data range (0.519±0.070), but the deviation from the control was within +/- 15%, it can be considered as a biological variability of the test system.