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Environmental fate & pathways

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:
2002-06-19 to 2002-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Principles of method if other than guideline:
In the CO2-evolution test, known concentrations of the test substance (corresponding to 10 and 20 mg carbon/L) as sole source of organic carbon are added to inoculated mineral medium. Degradation is followed for 28 days by trapping the produced CO2 in sodium hydroxide and titrating the residual amount of hydroxide in the traps. The amount of CO2 produced from the test substance (corrected for that derived from the blank inoculum) is expressed as a percentage of the theoretical CO2 evolution (ThCO2). Due to the low solubility of the test substance in water and the high volatility, the test substance was directly applied on a glass fibre filter carrier according to the international standard ISO 10634 [5] for introduction into the test system.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source: oxidation ditch in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic sewage.
- Treatment given: The medium was aerated with CO2-free air over night before use. The original sludge was mixed to provide homogeneity, and 278 mL of the sludge was used to inoculate 50 L medium to yield a final inoculum concentration of about 30 mg/L solid substance.
Duration of test (contact time):
28 d
Initial conc.:
14.64 mg/L
Based on:
test mat.
Initial conc.:
28.82 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Additional substrate: 5mL 0.6 or 0.4 M NaOH
- Test temperature: 20 +/- 2 °C
- pH: 7.38 - 7.49
- Continuous darkness: yes
- Concentration: The final (nominal) test concentrations of 14.64 and 28.82 mg/L were prepared by applying 16.5 and 33.0 µL on to glass fibre filters (Whatman GF/C, 0 47 mm). The filters were added separately to 1L inoculated medium in six separate bottles for each test concentration. This resulted in mean final concentrations of 10.2 and 20.2 mg C/L.
- Acidification: On day 14 and day 28, 1mL concentrated HCl was added to 3 of the blank control bottles, 2 of the inoculum activity bottles and 3 bottles of each of the 2 test substance series, to remove the remaining dissolved CO2 in the medium. The CO2 evolved after acidification was trapped during a period of approximately 1 day. On day 28, concentrated HCI was also added to both toxicity controls.

CONTROL AND BLANK SYSTEM
- Blank control: 6 control bottles with untreated glass fibre filters in 1L inoculated medium. 3 bottles were acidified after 14 days of incubation.
- Inoculum activity control: 68.0 mg of reference substance sodium acetate was added to 4 bottles containing 1L inoculated medium and untreated glass fibre filter carriers. Final test concentration of substance sodium acetate was achieved by dissolving 6.8015 g in ultrapure water and filled up to 100 mL with ultrapure water. From this stock solution 1 mL volumes were added to one litre of inoculated medium.
- Inoculum toxicity control: sodium acetate (68.0 mg/L) was also added to further duplicate bottles containing 29.32 mg/L of test substance.

Reference substance:
acetic acid, sodium salt
Remarks:
Purity: 99%
Key result
Parameter:
% degradation (CO2 evolution)
Value:
91.8
Sampling time:
28 d
Details on results:
The maximum mean biodegradation of the test item was 91.8 % at the high concentration (20.2 mg C/L) after acidification.
The 60% degradation level was reached (based on acidified samples) within 28 days, and also the 10d-window criterion. Therefore the test item was assessed to be readily degradable.

Inoculum activity and toxicity control

The biodegradation of sodium acetate in the control series (inoculum activity control) was used as a measure for the microbial activity of the inoculum. A reduction of the biodegradation of acetate in the presence of the test substance (toxicity control) would indicate an inhibiting effect on the inoculum activity.

The results show that sodium acetate was sufficiently degraded within two weeks. With the CO2 released upon acidification the inoculum activity control demonstrated a biodegradation of 85.0% after 14 days.

The percentage degradation of sodium acetate (68.0 mg/L) plus the test item (29.3 mg/L) in the toxicity control was calculated on the basis of the sum of the theoretical CO2-production of both substances. The CO2 -production was much higher in the toxicity control than that in the activity control. This means that the test item is not toxic to the inoculum, and it is degraded under these conditions. The test substance was also not toxic based on the percentage degradation, which was higher than 25%.

Results of biodegradation test with test item

Days

Test item (mg/L)

14.6

28.8

mg CO2

% ThCO2

mg CO2

% ThCO2

7

23.8

63.8

45.0

60.7

14

28.5

76.2

55.7

75.1

21

31.5

88.3

59.3

84.6

28

31.7

90.2

60.7

89.7

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance was readily biodegradable. The maximum degree of biodegradation was 91.8% of a concentration of approximately 20 mg C/L after 28 days of incubation. It also passed the 10-day-window criteria.
The substance was not toxic to the inoculum.
Executive summary:

The objective of the current study was to determine the ready biodegradability of the test substance in medium inoculated with activated sludge by measuring the CO2 production during 28 days of incubation.

The biodegradability was determined by a modified method according to the OECD Guideline 301: Ready Biodegradability, 301B CO2 Evolution Test and EEC Directive 92/69/EEG Part C, method C.4-C, using a modified CO2 trapping system. The study was carried out in conformity with the OECD principles of Good Laboratory Practice.

The CO2-evolution biodegradability test was carried out in closed 2L volume glass bottles containing 1L inoculated medium. A vial suspended from the screw-cap of each bottle contained a CO2 absorbing fluid. The vials containing CO2 absorbing fluid were replaced at weekly intervals and the trapped CO2 was determined by titration. Activated sludge taken from an oxidation ditch used to treat domestic sewage was used as inoculum. The inoculum concentration was 30 mg (dry weight)/L.

The test item was introduced into the inoculated medium by use of glass fibre filter carriers. Due to the high volatility of the test substance the glass fibre filters were weighted before they were added to the inoculum. Two concentrations of about 14.64 and 28.82 mg/L, which corresponds to 10.2 and 20.2 mg carbon/L, respectively, were tested with 6 bottles for each test concentration. The test was completed with a control blank with inoculated medium only, a control an inoculum activity with sodium acetate as reference substance, and a toxicity control with sodium acetate and 29.3 mg/L test item.

The inoculum activity and toxicity control tests with sodium acetate showed that the activity in the toxicity control was much higher than in the activity control, and that the test substance did not inhibit the degradation of the reference substance. This means that the test substance is not toxic to the inoculated medium.

The test item degraded for more than 90% at both concentrations after 28 days of incubation. The criteria given in the guideline (i.e. > 60% degradation reached within a 10 day period counting from the day that the level of degradation exceeded 10%) were met. Therefore, the test item is assessed to be readily biodegradable in this test.

Description of key information

readily biodegradable (91.8 % after 28 days; OECD 301 B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

For this endpoint there is one studies available. This study was performed according to OECD 301B: CO2 evolution test, using a modified CO2 trapping system. In this experiment the test item was administered to the test system via a glass fibre filter carrier. This is in line with the instructions applicable to volatile substances with a low water solubility. The test item degraded more than 90% with the 28 days and met the 10 -day window criteria. The test item is hence considered to be readily biodegradable. This study is assigned as key study for this endpoint.