Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
: A settled sludge was used instead of a centifuged sludge.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test solutions

Details on test solutions:
Stock solution:
200.6 mg test item were mixed and made up to 1000 ml with water.
The stock solution (suspension) was freshly prepared at the start of the test.


Test Concentrations:
Test item: 100.0, 32.0, 10.0, 3.2 and 1.0 mg/L
Reference item: 32.0, 10.0 and 3.2 mg/L
Calculated amounts of test item to produce the desired test concentrations were mixed with 114 ml water and 6.4 ml nutrient solution
(= test solution). Then 80 ml of the sludge were added. The test item, 6.4 ml of the nutrient solution, 114 ml water and 80 ml of the sludge were mixed in 250 ml BOD flasks and aerated immediately.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Inoculum:
Activated sludge collected from the sewage treatment plant of CH-4153 Reinach on February 2, 2000.
The pH after collection was 7.8.
The preparation was carried out according to the method described in the guideline.
The sludge was separated from the aqueous layer by settling instead of centrifugation. The pH of the sludge before use was 7.8.
The sludge concentration in the test bottles was 1.66 g suspended solids/L.

Nutrient Solution:
16g Peptone
11g Meat extract
3.0g Urea
0.7g NaCL
0.4g CaCl2.2H2O
0.2g MgSO4.7H2O
2.8g K2HPO4
Were dissolved and the volume made up to one litre with de-chlorinated drinking water.

Study design

Test type:
static
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
20 ± 2 °C
pH:
The pH after collection was 7.8.
The pH of the sludge before use was 7.8.
The pH of the test mixtures at the end of exposure was 8.6 - 8.8
Nominal and measured concentrations:
The sludge concentration in the test bottles was 1.66 g suspended solids/L.
Test Concentrations:
Test item: 100.0, 32.0, 10.0, 3.2 and 1.0 mg/L
Reference item: 32.0, 10.0 and 3.2 mg/L
Details on test conditions:
TEST SYSTEM
Vessels: 250 ml BOD flasks with gas inlet
Water: De-chlorinated drinking water
Temperature: 20 ± 2 °C
Duration: 3 hours.

Measurements:
Oxygen consumption per hour in mg/L with a ORION – Electrode Type 97-08, ORION Microprocessor Ionalizer 901 and plotted on a recorder.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
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:
other: EC20
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:
other: EC80
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Effect Concentrations:
The inhibitory effect of the test item was expressed as a percentage of the mean respiration rates of two controls. Following values were calculated based on nominal concentrations:
The test item was tested at nominal concentrations of 1.0, 3.2, 10.0, 32.0 and 100 mg test item/L after a contact time of 3 hours. The calculation of the following effect concentrations was based on these nominal values.

EC50 (3h) : > 100 mg/L

EC20 (3h) : > 100 mg/L
EC80 (3h) : > 100 mg/L

Conclusion:
The EC50 (3h) for CA 2218 A is > 100 mg/L. Based on the inherent toxicity of CA 2218 A in this test alone, the test item can be classified as:
“ not toxic to bacteria”.
Results with reference substance (positive control):
For the reference item (tested nominal concentrations) 3.2, 10 and 32 mg/L:

EC50 (3h) : > 13.9 mg/L

EC20 (3h) : > 4.3 mg/L
EC80 (3h) : > 45.0 mg/L


Test Validity:
The deviation of the respiration rates in controls was 12 %. The EC 50 (3h) of the reference item was 13.9 mg/L. Since the deviation of the
respiration rates of the controls was < 15 % and the EC 50 (3h) of the reference item was in the range of 5 to 30 mg/L, the test was valid in terms of
the given validity criteria.
Reported statistics and error estimates:
The inhibitory values were calculated on the basisof the measured time pnded oxygen comsumption of a blank and test solution. The results were
determined after calculation of the linear regression.

Any other information on results incl. tables

 

Sample

Concentration

(mg/L)

Consumption Rate (mg/L/h)

Inhibition (%)

pH

Blank 1

Blank 2

Reference 1

Reference 2

Reference 3

0.0

0.0

32.0

10.0

3.2

40.2

35.4

11.4

20.9

33.7

-6

6

70

45

11

8.8

8.7

8.8

8.8

8.7

Test conc. 1

Test conc. 2

Test conc. 3

Test conc. 4

Test conc. 5

100.0

32.0

10.0

3.2

1.0

31.8

33.3

33.8

35.3

35.2

16

12

10

7

7

8.7

8.6

8.7

8.8

8.8

 

The pH was measured after 3 h aeration.

Negative inhibition results, when obtained in a test, are a consequence of the comparison of oxygen consumption values obtained in the blank, and the higher oxygenconsumption vlaues obtained in the presence of the test item, which are due to biological variations between the various test samples.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EC50 (3h) for CA 2218 in this test alone, the test item can be classified as: “ not toxic to bacteria”.
Executive summary:

 

The respiration rate of an activated sludge fed with standard amount of synthetic sewage feed was measured after a contact time of 3 hours. The respiration rate of the same activated sludge was also measured in the presence of 1.0, 3.2, 10.0, 32.0 and 100 mg test item/L and 3.2, 10 and 32 mg/L reference item, respectively. The sludge concentration in the test bottles was 1.66 g suspended solids/L. 

 

The test with CA 2218 A was conducted at a temperature of 20 ± 1°C. The pH of sludge was 7.8 after collection and 8.7 after preparation (before use). The pH of the mixtures in the test bottles was 8.6 – 8.8 at the end of exposure time. 

The EC50 (3h) of the reference item 3,5-Dichlorophenol was 13.9 mg/L

 

The inhibitory effect of the test item was expressed as a percentage of the mean respiration rates of two controls. Following values were calculated based on nominal concentrations:

 

EC50 (3h) : > 100 mg/L

 

EC20 (3h) : > 100 mg/L

EC80 (3h) : > 100 mg/L

 

Conclusion

The EC50 (3h) for CA 2218 in this test alone, the test item can be classified as: “ not toxic to bacteria”.