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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2011-09-19 to 2012-05-10
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
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
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.

Before the start of the test, defined amounts of the test item stock solution (2500 mg/L) were dosed into the test flasks containing synthetic sewage by mechanical dispersion. The respective flasks were filled with the appropriate amount of deionized water to give the selected test 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.
Vehicle:
yes
Details on test solutions:
FORMULATION

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 (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 7.64. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period.
Hardness:
No data.
Test temperature:
20.1 – 21.0 degrees C (during the incubation) and
19.1 – 20.6 degrees C (during oxygen measurement)
pH:
7.40 to 7.66
Dissolved oxygen:
8.3 to 8.5 mg O2/L
Salinity:
Not applicable.
Nominal and measured concentrations:
The test concentrations (10, 31, 100, 313 and 1000 mg/L). 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: 20.1 – 21.0 degrees C (during the incubation) and 19.1 – 20.6 degrees 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.

PREPARATION 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 volume of the stock solution 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).

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 7.77 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
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 was inhibited between 7.7% and 12.8% in the examined test concentration range of 10-1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/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 35.9% 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 66.7% and 79.5%, respectively.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 8.52 mg/L with 95% confidence limits of 6.55 to 11.08 mg/L.
Reported statistics and error estimates:
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 8.52 mg/L with 95% confidence limits of 6.55 to 11.08 mg/L.

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

0.380

7.68

7.77

8.4

8.1

10

C2

Control

0.400

7.55

7.66

8.4

8.5

 

Mean

0.390

 

Deviation (%)

5.15

5

T1

Test item

10

0.360

7.7

7.48

7.64

8.5

8.3

6

T2

Test item

31

0.360

7.7

7.40

7.66

8.5

8.3

7

T3

Test item

100

0.350

10.3

7.51

7.64

8.4

8.3

8

T4

Test item

313

0.350

10.3

7.53

7.60

8.5

8.4

9

T5

Test item

1000

0.340

12.8

7.60

7.60

8.4

8.3

* 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

0.380

7.68

7.77

8.4

8.1

10

C2

Control

0.400

7.55

7.66

8.4

8.5

 

Mean

0.390

 

Deviation (%)

5.15

2

R1

Ref. item

5

0.250

35.9

7.55

7.60

8.4

9.0

3

R2

Ref. item

16

0.130

66.7

7.47

7.78

8.2

8.7

4

R3

Ref. item

32

0.080

79.5

7.41

7.61

8.0

8.9

* start and end of 3-hour aeration

ID:  Code of each group

Validity criteria fulfilled:
yes
Conclusions:
A laboratory test was carried out with Reactive Yellow F01-0555 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.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 7.7% and 12.8% in the examined test concentration range of 10-1000 mg/L, which was considered 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 8.52 mg/L with 95% confidence limits of 6.55 to 11.08 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.
Executive summary:

A laboratory test was carried out with Reactive Yellow F01-0555 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.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 7.7 % and 12.8 % in the examined test concentration range of 10-1000 mg/L, which was considered 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 8.52 mg/L with 95% confidence limits of 6.55 to 11.08 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.

Description of key information

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

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information