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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 October 2003 to 10 October 2003
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal 500 mg/L stock solution of the test material was prepared in deionised water. This stock was observed to be a clear, pale yellow solution after 60 minutes stirring and 30 minutes ultrasonic treatment and the pH was measured as 4.8. Test solutions were prepared by the addition of a known volume of this stock solution to a total volume of 500 mL aerated tap water, synthetic sewage and activated sludge.
- Controls: Two culture control flasks.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Method of cultivation: Activated sludge was obtained from Buckland Sewage Treatment Works, Newton Abbot, Devon, UK, 3 days prior to testing. The activated sludge was settled and the concentrated sludge was washed with tap water. The sludge was fed with 50 mL OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature.

A synthetic sewage mixture was prepared containing the following per litre of deionised water:
15.2 g peptone
10.5 g meat extract
2.9 g of urea
0.7 g of NaCl
0.4 g of CaCl2.2H2O
0.2 g of MgSO4.7H2O
2.8 g of H2HPO4

- Total filterable solids: 7515 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
6.1
Dissolved oxygen:
At least 6.5 mg O2/L at the end of the 3 hour aeration period
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Flask
- Aeration: The flasks were aerated throughout the exposure period
- No. of vessels per control (replicates): Duplicate
- No. of vessels per concentration (replicates): Duplicate
- Final solids concentration: 1600 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted with hydrochloric acid

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate of each culture was measured after 3 hours and compared with the mean respiration rate of the two control cultures. The rate of oxygen uptake was measured in a confined perspex cell into which a polargraphic oxygen electrode was inserted.The electrode was connected to a meter whose output was recorded on a potentiometric chart recorded. The dissolved oxygen concentration after the 3 hour period was at least 6.5 mg O2/L. The respiration rate was measured over the linear part of the curve for five to ten minutes. The rates for oxygen uptake were expressed at mg O2/L/h. The respiration rates of the flasks dosed with the test substance of the reference substance were expressed as percentages of the mean respiration rate of the control flasks, from which the percentage inhibition was derived:

% inhibition = (1-(Respiration rate of test flask/Mean respiration rate of control flasks)) x 100
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? The results are valid
- Relevant effect levels: The reference substance caused substantial inhibition of the respiration rate of the activated sludge, From the results obtained, the 3 hour EC50 was estimated to be 4.8 mg/L. This is slightly less that the expected normal range of 5 to 30 mg/L indicating that the sludge was more sensitive than usual. As no inhibition was observed for the test substance , this is not considered to have affected the validity of the study.

Table 2: Inhibition results

Test Substance

Nominal Test Concentration (mg/L)

pH

Respiration rate (mg O2/L/h)

Percentage Mean Control Respiration Ratea

Percentage Inhibition

Start

End

Control

Control

6.9

7.7

27.5

-

-

3,5-DCPb

100

7.2

7.7

0

0

100

3,5-DCP

32

7

7.7

2

7

93

3,5-DCP

10

6.9

7.8

8

28

72

3,5-DCP

3.2

6.9

7.9

18

63

38

3,5-DCP

1

7

7.8

20.5

71

29

Test material

100

6.9

7.6

27

94

<10

Test material

32

6.9

7.6

29.5

102

<10

Test material

10

6.9

7.7

29.5

102

<10

Test material

3.2

6.9

7.7

31.5

109

<10

Test material

1

7

7.7

30

104

<10

Control

Control

7

7.7

30

-

-

aMean control respiration rate = 28.8 mg O2/L/h

b3,5-dichlorophenol

Validity criteria fulfilled:
not specified
Remarks:
The information on the dissolved oxygen concentration is not sufficient to assess the study against the criteria provided in the OECD guideline.
Conclusions:
Under the conditions of the test, <10 % inhibition was observed in all the tested concentrations. The 3 h EC50 of the test material was determined to be >100 mg/L. The 10 % effect concentration (EC10) is defined as the concentration resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentage inhibition less than or equal to 10 % was within the expected experimental variability of the test and was considered not to be an effect of the test material. The NOEC under the conditions of this study is therefore equivalent to the EC10 in this study and was determined to be 100 mg/L. The study is considered to be reliable, relevant and adequate for risk assessment purposes.
Executive summary:

The effect of the test material on the respiration rate of activated sludge was investigated in accordance with the standardised guidelines OECD 209 and EU Method C.11. Activated sludge was exposed to nominal concentrations of the test material of 1.0, 3.2, 10, 32 and 100 mg/L with controls and a reference substance (3,5-dichlorophenol) run concurrently for comparison. Less than 10 % inhibition was observed in all the tested concentrations and the 3 hour EC50 was determined to be >100 mg/L. The 10 % effect concentration (EC10) is defined as the concentration resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentage inhibition less than or equal to 10 % was within the expected experimental variability of the test and was considered not to be an effect of the test material. The NOEC under the conditions of this study is therefore equivalent to the EC 10 in this test was determined to be 100 mg/L.

Description of key information

3 h EC50 = > 100 mg/L, NOEC = 100 mg/L, OECD 209, Bowles & Maynard 2003

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The effect of the test material on the respiration rate of activated sludge was investigated in accordance with the standardised guideline OECD 209. Activated sludge was exposed to nominal concentrations of the test material of 1.0, 3.2, 10, 32 and 100 mg/L with controls and a reference substance (3,5-dichlorophenol) run concurrently for comparison. Less than 10% inhibition was observed in all the tested concentrations and the 3 hour EC50 was determined to be >100 mg/L. The 10% effect concentration (EC10) is defined as the concentration resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentage inhibition less than or equal to 10 % was within the expected experimental variability of the test and was considered not to be an effect of the test material. The NOEC under the conditions of this study is therefore equivalent to the EC 10 in this test was determined to be 100 mg/L. The available data is considered to be complete and the result determined, 3 h EC50 value ≥100 mg/L, was taken forward for risk assessment.