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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:
From 30 October 2019 to 30 December 2019
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)
Deviations:
yes
Remarks:
Minor deviation: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound)
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
yes
Remarks:
Minor deviation: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound)
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)
Deviations:
not specified
GLP compliance:
no
Remarks:
Inspected on 12-13 November 2019 / Signed on 09 December 2019
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (24-10-2019) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter was dried for 1.5 hour at 102.7 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.9 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated by stirring on a magnetic stirrer plate at ambient temperature (± 21 °C) for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
60 d
Initial conc.:
2 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: Deionized water used in the Closed Bottle test contained per liter of water 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4.2H2O, 22.50 mg MgSO4·7H2O, 27,51 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 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, 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L.

- Test procedures: Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel (0.2 g silica gel / bottle), 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L 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 at day 42 and 60 using the bottles of day 28 and 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).

- Analyses: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger
Reference substance:
acetic acid, sodium salt
Preliminary study:
The theoretical oxygen demand (ThOD) of the test item used to calculate the biodegradation percentages is 2.98 g oxygen/g test substance. This ThOD was calculated with the molecular formula and by assuming that the ThOD of all constituents is equal to the ThOD of the known constituents. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.
Test performance:
The validity of the test is demonstrated by an endogenous respiration of 1.2 mg/L at day 28. Furthermore, the differences of the replicate values at day 60 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80. 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:
40
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
60 d
Remarks on result:
other: ultimately (completely) biodegradable
Details on results:
- 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 the test substances 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.
- Test conditions: The pH of the media was 7.4 (control, control with silica gel, test and reference) at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 7.2. The pH
of the medium for the control, control with silica gel, and test bottles at day 60 was 7.2. The temperature ranged from 22.6 to 22.9 °C which is within the prescribed temperature range of 22 to 24°C.

Table 5.2.1/1: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in Closed Bottle test

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

 

Test substance

Acetate

Test substance

Acetate

0

0.00

0.00

0

0

7

0.25

3.70

4

71

14

1.15

4.20

19

80

21

1.20

 

20

 

28

2.40

 

40

 

42

3.45

 

58

 

60

3.70

 

62

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Remarks:
Not readily biodegradable but ultimately (completely) biodegradable, and therefore not persistent.
Conclusions:
The test substance was biodegraded by 40% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 62% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable and allows classification of the test item as not persistent.
Executive summary:

To assess the biotic degradation of the test substance, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The test item (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.6 to 22.9 °C for 60 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The test substance was biodegraded by 40% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 62% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60-day test period allows classification of the test item as not persistent. The test is valid as shown by an endogenous respiration of 1.20 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 80% 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.

Description of key information

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

40% biodegradation after 28 days and 62% biodegradation after 60 days

Not readily biodegradable but ultimately (completely) biodegradable, and therefore not persistent.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information

One experimental key GLP study is available to assess the biodegradation potentiel of the registered substance.


This valid Closed Bottle test (Nouryon, 2020) was performed according to slightly modified OECD, EU and ISO Test Guidelines, with GLP compliance. The registered substance (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.6 to 22.9 °C for 60 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. According to the results of this study, the test substance did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The test substance was biodegraded by 40% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 62% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60-day test period allows classification of the test item as not persistent.


Another experimental study (Firmenich, 2021) was included in the dossier and presents better biodegradation results for the registered substance :


Two out of four tests (test 1 and 3) delivered results fulfilling the criteria for the test material to be considered as readily biodegradable under the strict terms and conditions of OECD Guideline N° 301F. The other two tests (test 2 and 4) demonstrate that the test substance is not persistent but ultimately (completely) biodegradable according to ECHA (European Chemicals Agency, ECHA guidance chR7b, p179&198, ”Enhanced biodegradation test”). The study demonstrates therefore the potential for the test material to be readily biodegradable. However, as this study was not performed under GLP compliance, this conclusion cannot be used as key result and this study is only assessed as another information (adequacy of study).