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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:
19 April 2002 to 20 June 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
Additional NaNo3 was added to the mineral medium to prevent nitrogen limitation to occur
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
yes
Remarks:
Additional NaNo3 was added to the mineral medium to prevent nitrogen limitation to occur
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge ws taken from an oxidation ditch situation in the municipality of Hazerswoude, The Netherlands on May 14, 2002.
- Concentration of sludge: 2.05 mL
Duration of test (contact time):
35 d
Initial conc.:
70 mg/L
Based on:
test mat.
Details on study design:
Preparation of the biodegradation test flasks:
The test material was added to the test systems on an inert carrier according to the International Standard ISO 10634. The final test concentrations of test material were prepared by dissolving 0.8402 g of the test material in 10 mL ethanol (Stock 1). An aliquot of 250 µL of Stock 1 was applied to glass fibre filters (Whatman GF/C, 0 47 mm). After being air-dried, a glass fibre filter was placed in each test flask and subsequently the flasks were filled with 300 mL mineral medium. This resulted in the final concentration of 70.0 mg/L. This series was completed with an inoculum blank containing mineral medium and an ethanol treated fibre filter.

Preparation of the activity control:
The final test concentration of sodium acetate was prepared by dissolving 2.9930 g of the reference substance in 100 mL of ultrapure water. From this stock solution 1 mL was added to 300 mL mineral medium resulting in a final nominal test concentration of 100 mg/L sodium acetate.

Preparation of the toxicity control:
The test material was added to the test flasks as described for the biodegradation flasks. In addition 1 mL of the reference substance stock solution was added.

Test series:
Test material - Concentrations = 0 (with filter), 70 mg/L; Number of replicate flasks = 3; Oxygen concentration determined = every 4 hours
Inoculum activity control - Concentration of the reference substance = 0 (without filter), 100 mg/L; Number of replicate flasks = 3 (blanks) and 2; Oxygen concentration determined = every 4 hours
Toxicity control - Concentration of the reference substance = 70 mg/L; Concentration of the test substance = 100 mg/L; Number of replicate flasks = 2; Oxygen concentration determined = every 4 hours

pH measurement:
After addition of the test material and mineral medium, the pH was measured in each treatment before inoculation, except for the blanks. If necessary, the pH was adjusted to 7.4 +/-0.2 with 0.1 M HCl or 0.4 M NaOH solution. The pH was also measured in the test media at the end of the test.

Inoculation, incubation and measurements:
After measurement of the pH and its adjustment, the filled flasks were inoculated with 2.05 mL of the diluted sludge. This resulted in a concentration of 30 mg (dry weight)/L mineral medium. The flasks were closed and placed in the incubator of the Manometric Respirator (Micro-Oxymax). When all test flasks were placed in the incubator and connected to the Micro-Oxymax, the incubator was closed and the oxygen and temperature measurements were started.

Operation of the Micro-Oxymax:
The Micro-Oxymax measures the percentage oxygen in the air of the respective flasks and calculated, based on the earlier measurement, the resulting oxygen consumption in a certain time frame. Based on these values the oxygen consumption per flasks can be derived.

Termination of the experiment:
After 28 days of incubation it was decided to prolong the test by one week because the oxygen consumption had not reached the plateau phase. The actual termination was after 35 days (830 h). After termination of the test, the pH was measured in each flask.
Reference substance:
acetic acid, sodium salt
Test performance:
pH of medium in test flasks: 7.3-7.4 after 35 days
The pH of at the end of the reference and toxicity test was 8.0
Average temperature in flasks was 23 +/- 0.5 ºC during the test
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
TEST MATERIAL:
- ThOD(NH3): 0.71 mg O2/mg test material.
- BOD (mg O2/mg test material): -0.01 (14d), -0.02 (28d) and -0.02 (35d).
- Biodegradation: -1% (14d), -3% (28d), -3% (35d).

CONTROLS:
- Cumulative oxygen consumption (inoculum blank, with filter): 3.7 mg O2/flask (14d), 5.1 mg O2/flask (28d), 5.5 mg O2/flask (35d).
- Cumulative oxygen consumption (inoculum blank, without filter): 2.4 mg O2/flask (14d), 3.2 mg O2/flask (28d), 3.5 mg O2/flask (35d).
- Cumulative oxygen consumption (inoculum activity control): 19.0 mg O2/flask (14d), 21.0 mg O2/flask (28d), 21.4 mg O2/flask (35d). The values are corrected for the blank with filter.
- Biodegradation (inoculum activity control): 81% (14d), 86% (28d), 87% (35d).
- Cumulative oxygen consumption (toxicity control, without filter): 21.0 mg O2/flask (14d), 23.7 mg O2/flask (28d), 24.1 mg O2/flask (35d). The values are corrected for the blanks without filter.
- Biodegradation (toxicity control): 49% (14 d), 53% (28 d), 53% (35 d).

OTHER: The cumulative oxygen consumption in the toxicity control (sodium acetate and test substance) after 14 days was slightly higher than that of the inoculum activity control (sodium acetate) alone. This indicated that the test material did not inhibit the degradation of sodium acetate at the concentration tested. Based on the combined ThOD of both substances a biodegradtion of >25% was reached which, according to the guidelines, means that the test material is considered not toxic to the inoculum.

After 28 days the oxygen consumption that could be attributed to the degradation of the test material in the toxicity control was 0.8 mg O2/flask. This corresponds to an oxygen consumption of 2.7 mg O2/L and compared with the ThOD (NH3) of the material, a biodegradation of 5.4% was calculated. This is higher than observed in the test material sample itself (-0.02%) and may indicate that some biodegradation of the test material may occur under appropriate conditions.

The test material was biodegraded 0% after 28 days at a test concentration of 70 mg/L and is therefore considered to be not readily biodegradable. However, the results from the toxicity control indicate that some biodegradation may occur under appropriate conditions.

Kinetic of test material (in %):
= 0 after 14 day(s)
= 0 after 28 day(s)
= 0 after 35 day(s)
Kinetic of control substance (in %):
= 81 after 14 day(s)
= 87 after 35 day(s)
Degradation products: not measured
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material did not biodegrade in a manometric respiration test at a test concentration of 70 mg/L after 35 days incubation. Based on these results, the test material is considered to be not readily biodegradable.
Executive summary:

The biodegradability of the test material was determined as described in the OECD Guideline 301F, using oxygen consumption as test criterion in a 35 day test. This method is in agreement with EU Guidelines C.4 -D. The only deviation to the guideline is that additional NaNO3 was added to the mineral medium to previent nitrogen limitation occurring.

One concentration of 70 mg/L was tested corresponding to a ThODNH3 of 49.7 O2/l. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (30 mg (d.w.)/l).

The test fulfilled the conditions of validity given by the guidelines. The inoculum activity appeared to be sufficient; the reference substance sodium acetate reached the 60% pass level of degradation within 14 days. In a toxicity control test with 70 mg/L of test material and 100 mg/L sodium acetate, no inhibition of the degradation of sodium acetate was found.

The test was prolonged to 35 days because the plateau phase had not been reached after 28 days of incubation. The test material was not degraded. The 60% degradation criteria was not met and therefore the test material was not considered readily biodegradable.

A 5.4% biodegradation of the test material in the toxicity control may indicate that it may degrade under appropriate conditions.

Description of key information

The test material did not biodegrade in a manometric respiration test at a test concentration of 70 mg/L after 35 days incubation. Based on these results, the test material is considered to be 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 biodegradability of the test material was determined as described in the OECD Guideline 301F, using oxygen consumption as test criterion in a 35 day test. This method is in agreement with EU Guidelines C.4 -D. The only deviation to the guideline is that additional NaNO3was added to the mineral medium to previent nitrogen limitation occurring.

One concentration of 70 mg/L was tested corresponding to a ThODNH3of 49.7 O2/l. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (30 mg (d.w.)/l).

The test fulfilled the conditions of validity given by the guidelines. The inoculum activity appeared to be sufficient; the reference substance sodium acetate reached the 60% pass level of degradation within 14 days. In a toxicity control test with 70 mg/L of test material and 100 mg/L sodium acetate, no inhibition of the degradation of sodium acetate was found.

The test was prolonged to 35 days because the plateau phase had not been reached after 28 days of incubation. The test material was not degraded. The 60% degradation criteria was not met and therefore the test material was not considered readily biodegradable.

A 5.4% biodegradation of the test material in the toxicity control may indicate that it may degrade under appropriate conditions.