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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:
2016-09-27 to 2016-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Akzo Nobel NV, Environmental Chemistry, Zutphenseweg 10, 7418 AJ Deventer, The Netherlands
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (22-09-2016) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L (van Ginkel and Stroo, 1992). The inoculum was not pre-exposed to the test substance.
Duration of test (contact time):
60 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers

- Nutrients, and stocks: The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top and the content was mixed vigorously. Subsequently 0.10 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 1.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L

- Test procedures: The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing silica gel and inoculum, 10 bottles containing inoculum, silica gel, and test substance, and 6 bottles containing sodium acetate and inoculum. The concentrations of the test substance, and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

- Test conditions: The pH of the media was 7.3 at the start of the test. The pH of the media at day 28 was also 7.3 (test and controls). Temperatures were within the prescribed temperature range of 22 to 24°C.
Reference substance:
acetic acid, sodium salt
Remarks:
Purity >99%
Test performance:
The validity of the test is demonstrated by an endogenous respiration of 0.9 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
0 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
7 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
14 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
21 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
12
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
15
Sampling time:
42 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
35
Sampling time:
60 d
Details on results:
- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of Reseda Body (mono-constituent) is 2.6 mg/mg. The ThOD of sodium acetate is 0.8 mg/mg.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of Reseda Body (mono-constituent) to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.
- Biodegradability: Reseda Body (mono-constituent) was biodegraded by 12% at day 28 in the Closed Bottle test (Table II). In the prolonged Closed Bottle test this test substance was biodegraded by 35% at day 60. The test item should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrates that this substance is partially biodegradable. A plateau of biodegradation was not reached at the end of the test indicating that the test item could be degraded completely in time. The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is partially degradable in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 81.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the test, the substance is biodegraded by 12% at day 28 and by 35% at day 60 in the prolonged Closed Bottle test. The test item should therefore not be classified as readily biodegradable.
Executive summary:

In order to assess the biodegradation of Reseda Body (mono-constituent), a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. 2.0 mg/L domestic, non-adapted, activated sludge was exposed to 1 mg/L test substance for 60 days. The test is valid as shown by an endogenous respiration of 0.9 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 81% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period. Under the conditions of the test, the substance is biodegraded by 12% at day 28 and by 35% at day 60 in the prolonged Closed Bottle test. The test item should therefore not be classified as readily biodegradable.

 

Description of key information

In order to assess the biodegradation of Reseda Body, a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. 2.0 mg/L domestic, non-adapted, activated sludge was exposed to 1 mg/L test substance for 60 days. The test is valid as shown by an endogenous respiration of 0.9 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 81% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period. Under the conditions of the test, the substance is biodegraded by 12% at day 28 and by 35% at day 60 in the prolonged Closed Bottle test. The test item should therefore not be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information