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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 24 to March 24, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301 B and EU Method C.4-C with GLP statement. No deviation was observed and all validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on September 22, 1999 / signed on January 18, 2000
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
INOCULUM
Type: Inoculum was collected from a water treatment plant containing effluent from a predominantly domestic origin.
Source: Water treatment plant of Evreux (27000 Evreux, France).
Preparation: The inoculum was prepared by initially sieving sewage sludge. The sludge was then centrifuged for 5 minutes, the supernatant was rejected and the pellet was redispersed in the mineral medium. The inoculum was preconditioned for 6 days before use. Air was bubbled through the inoculum during this preconditioning period.

PRECONDITION
Temperature: 22 ± 2 °C
Aeration: Air was bubbled through the inoculum until use.
Mineral medium: Reconstituted water
Duration of test (contact time):
28 d
Initial conc.:
10 other: mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: A quantity of 10 mL of solution A (KH2PO4, K2HPO4, Na2HPO4.2H2O and NH4Cl) was mixed with 800 mL water, then 1 mL of solutions B (CaCl2), C (MgSO4.7H2O) and D (FeCl3.6H2O and HCl) were added and made up to 1 litre with water. Mineral medium: Reconstituted water
- Solubilising agent (type and concentration if used): None
- Test temperature: 21-23 °C
- pH: 7.51 measured in the mineral medium before the start of the test; 8.00 to 8.80 measured in the test flasks at the end of the test.
- Aeration: Air was bubbled through each parallel at the rate of 30 - 100 mL/min during the test
- Suspended solids concentration: 12.0 mg/L (DW)
- Continuous darkness: Yes; The test was carried out in dark glass bottles fitted with dark glass stoppers and aeration tubes to reduce the quantity of light reaching the test suspensions.
- Culture homogeneity: All test suspensions were agitated using magnetic stirrers.
- Loading: Test vessels were loaded at 3 Litres of suspension per flask


TEST SYSTEM
- Culturing apparatus: Flasks
- Number of culture flasks/concentration: 2; two flasks containing the test substance (at 10.0 mg/L of TOC) and inoculum (test solutions)
- Method used to create aerobic conditions: The test flasks were aerated with CO2-scrubbed air overnight to purge the system of carbon dioxide and then attached in parallel to a series of three wash bottles filled with 100 mL of 0.0125 M barium hydroxide solution.
- Method used to create anaerobic conditions: Not applicable
OTHERS:
- The test and reference substances were added, as appropriate, to the flasks to give a test and reference substance concentrations of 10.0 mg of TOC per litre (37.1 mg of test item and 102.4 mg of sodium acetate). When all the substances had been added, the volume of the suspensions was made up to 3 litres. Carbon dioxide-scrubbed air was bubbled through the suspensions at the same rate for all preparations.
- Measurements of CO2 were made at the following times: Days 1, 4, 6, 8, 11, 14, 18, 21, 25 and 29. For each measurement, the first wash bottle nearest to the test flask was disconnected and titrated with 0.05 M HCl, using phenolphthalein as an indicator.
For calculation purpose, it was assumed that the volume necessary to titrate untitrated wash bottle would be the same as the volume needed to titrate 100 mL of the Ba(OH)2 stock solution. The remaining CO2 absorber bottles were connected to the test flasks so that the second wash bottle replaced the first one and an extra bottle containing fresh barium hydroxide solution was added to the far end of the series. Each time CO2 was analyzed, 100 mL of the barium hydroxide stock solution was titrated with the HCl solution in order to determine the maximum amount of acid required to titrate the wash bottles. On the 28th day, 1 mL of concentrated hydrochloric acid was added to each flask to stop the biodegradation and aerated overnight to drive off the remaining carbon dioxide. A final CO2 analysis of all wash bottles was made on the 29th day, this final analysis being representative of biodegradation of the 28th day.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; two flasks containing the inoculum (inoculum blanks)
- Procedure control: Yes with the reference substance; one flask containing the reference substance (sodium acetate at 10.0 mg/L of TOC) and inoculum (procedure control)
- Toxicity control: Yes; one flask containing the test substance (at 10.0 mg/L of TOC), the reference substance (sodium acetate at 10.0 mg/L of TOC) and inoculum (toxicity control)
Reference substance:
acetic acid, sodium salt
Preliminary study:
None
Test performance:
Three validity criteria were respected:
- Biodegradation in the reference test was 63% after 14 days then it was at least 60% within this period
- The blank value (average of the two controls) was ≤ 70 mg of CO2/L at the end of the test (52.8 mg/L)
- Biodegradation in the toxicity control was 31% (based on ThCO2) after 14 days then it was at least 25% within this period.

However, biodegradation values of test substance replicates deviated slightly by more than 20% (21.6%) at the end of the test. It was assumed that the difference between these biodegradation values was not sufficiently important to throw back into question this study. Such difference at the end of the test is by no means unusual when the biodegradation of the test substance is low because the precision of the method is lesser in this case.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
28 d
Details on results:
Biodegradation of the test substance totaled 7% over the test period.

No significant inhibition of the inoculum due to toxicity of the test substance was noted since the toxicity control reached 31% of the total organic carbon within 14 days. Biodegradation in this control totaled 40% after 28 days.

Points of degradation plot (test substance):
1.5 % degradation after 1 d
3.1 % degradation after 4 d
3.1 % degradation after 8 d
4.5 % degradation after 14 d
4.8 % degradation after 18 d
6.2 % degradation after 21 d
6.7 % degradation after 28 d

Results with reference substance:
Points of degradation plot (reference substance):
8.5 % degradation after 1 d
21.5 % degradation after 4 d
46.7 % degradation after 8 d
62.7 % degradation after 14 d
65.1 % degradation after 18 d
70.2 % degradation after 21 d
83.6 % degradation after 28 d

Table 5.2.1/1: Cumulative percentage of biodegradation

Day

Test substance

Reference substance

Toxicity control

No. 1

No. 2

Average

1

0.25

2.65

1.45

8.54

0.00

4

0.80

5.40

3.10

21.48

6.62

6

0.80

5.40

3.10

39.26

14.51

8

0.80

5.40

3.10

46.66

19.56

11

3.55

5.45

4.50

50.20

25.63

14

3.55

5.45

4.50

62.69

31.37

18

3.55

5.99

4.77

65.14

32.80

21

6.44

5.99

6.22

70.24

35.24

25

7.44

5.99

6.72

70.24

35.24

28

7.44

5.99

6.72

83.62

39.89

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Biodegradation of the test substance totaled 7% over the test period. Under the experimental conditions, the test substance is non-readily biodegradable in the 28-day modified Sturm test.
Executive summary:

The ready biodegradability of the test item was evaluated using a 28-day modified Sturm test according to the OECD Guideline No. 301 B, EU Method C.4 -C with GLP compliance.

The test substance was dissolved in reconstituted water (OECD mineral medium) prepared from deionised water with a conductivity < 10 µS/cm. Inoculum blanks, test solutions (test substance at 10.0 mg/L of TOC), procedure control [reference substance (sodium acetate at 10.0 mg/L of TOC) and inoculum] and toxicity controls (test substance (at 10.0 mg/L of TOC), reference substance (sodium acetate at 10.0 mg/L of TOC) and inoculum) were used. The inoculum consisted of sewage sludge sampled from the aeration tank of a sewage treatment plant and then aerated for 6 days. Inoculum concentration was 12.0 mg/L (dry weight) in all test vessels. CO2 scrubbed air was bubbled through the flasks over the test period. Test was performed at 21-23 °C and pH was7.51 to 8.80.

 

Biodegradation of the test substance totaled 7% (mean of two test flasks) over the test period. No significant inhibition of the inoculum due to toxicity of the test substance was noted since the toxicity control reached 31% of the total organic carbon within 14 days. Biodegradation in this control totaled 40% after 28 days. Degradation of sodium benzoate exceeds 60% after 14 days: the activity of the inoculum is thus verified.

 

Under the experimental conditions, the test substance is non-readily biodegradable in the 28-day modified Sturm test.

Description of key information

OECD Guideline 301B, GLP, key study, validity 1:

7% biodegradation after 28 days

Not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The ready biodegradability of the test item was evaluated using a 28-day modified Sturm test according to the OECD Guideline No. 301 B, EU Method C.4 -C with GLP compliance.

The test substance was dissolved in reconstituted water (OECD mineral medium) prepared from deionised water with a conductivity < 10 µS/cm. Inoculum blanks, test solutions (test substance at 10.0 mg/L of TOC), procedure control [reference substance (sodium acetate at 10.0 mg/L of TOC) and inoculum] and toxicity controls (test substance (at 10.0 mg/L of TOC), reference substance(sodium acetate at 10.0 mg/L of TOC) and inoculum) were used. The inoculum consisted of sewage sludge sampled from the aeration tank of a sewage treatment plant and then aerated for 6 days. Inoculum concentration was 12.0 mg/L (dry weight) in all test vessels. CO2scrubbed air was bubbled through the flasks over the test period. Test was performed at 21-23°C and pH was7.51 to 8.80.

 

Biodegradation of the test substance totaled 7% (mean of two test flasks) over the test period. No significant inhibition of the inoculum due to toxicity of the test substance was noted since the toxicity control reached 31% of the total organic carbon within 14 days. Biodegradation in this control totaled 40% after 28 days.Degradation of sodium benzoate exceeds 60% after 14 days: the activity of the inoculum is thus verified.

 

Under the experimental conditions, the test substance is non-readily biodegradable in the 28-day modified Sturm test.