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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:
14 Feb 2017 to 16 Mar 2017
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)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
1992
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)
Version / remarks:
1994
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (09-02-2017) 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. The inoculum was not pre-adapted to the test substance.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
as a percentage of ThOD
Details on study design:
TEST SYSTEM
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- pH: the pH of the media was 7.3 at the start of the test. The pH of the media at day 28 was 7.3 (test, control and control with silica gel)
- Test temperature: 22 to 24 °C.

PREPARATION AND MEDIA
- Deionized water: deionized water containing no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification system.
- Media: 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.
- Addition of test substance: procedure: 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 0.20 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 a 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, 10 bottles containing inoculum and silica gel with test substance, 6 bottles with inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 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.

SAMPLING
- Sampling frequency: two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.
- Parameters measured: the dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW) and meter (WTW). The pH was measured using a Eutech Cyberscan pH11 pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

CALCULATIONS OF THE RESULTS
- Calculation of the biodegradation percentages: The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Reference substance:
acetic acid, sodium salt
Remarks:
6.7 mg/L
Test performance:
VALIDITY
The validity of the test is demonstrated by an endogenous respiration of 0.8 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 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:
71
Sampling time:
28 d
Details on results:
Cyclemax (mono-constituent) was biodegraded by 71% at day 28 in the Closed Bottle test (see 'Any other information on results incl. tables'). Over 60% biodegradation was achieved in a period of approximately 13 days immediately following the attainment of 10% biodegradation at day 7 (see Illustration). Cyclemax therefore fulfilled the 14-day time window.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80.

Table: Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)

Oxygen concentration (mg/L)

Ocs

Ot

Oc

Oa

0

8.7

8.7

8.7

8.7

 

8.7

8.7

8.7

8.7

Mean (M)

8.7

8.7

8.7

8.7

7

8.2

7.5

8.2

4.0

 

8.2

7.6

8.2

4.1

Mean (M)

8.2

7.6

8.2

4.1

14

8.0

6.6

7.9

3.7

 

8.0

6.2

8.0

3.7

Mean (M)

8.0

6.4

8.0

3.7

21

7.8

4.5

7.9

 

 

7.9

4.2

7.9

 

Mean (M)

7.9

4.4

7.9

 

28

7.8

3.9

7.9

 

 

7.8

3.7

7.9

 

Mean (M)

7.8

3.8

7.9

 

Ocs: Mineral salts medium, inoculum and silica gel.

Ot: Mineral salts medium, inoculum, test material (2.0 mg/L) and silica gel.

Oc: Mineral salts medium, and inoculum

Oa: Mineral salts medium, inoculum and sodium acetate (6.7 mg/L).

 

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

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

0

0.0

0.0

0

0

7

0.6

4.1

11

76

14

1.6

4.3

29

80

21

3.5

 

63

 

28

4.0

 

71

 

 

Validity criteria fulfilled:
yes
Remarks:
see 'Test performance'.
Interpretation of results:
readily biodegradable
Conclusions:
The substance is biodegraded by 71% at day 28 in the OECD TG 301D Closed Bottle Test and the 14-day time window criterion was met. Therefore, the substance should be classified as readily biodegradable.
Executive summary:

The biodegradation potential of the substance in water was determined in a screening study according to OECD TG 301D (Closed Bottle Test) and in compliance with GLP criteria. In this study 2.0 mg/L test substance was inoculated with secondary activated sludge of a city sewage treatment plant for 28 days under aerobic conditions in the dark. After the 28-day incubation period, the cumulative percentage biodegradation was calculated from the oxygen depletion (BOD/ThOD ratio). After the 28-day incubation period 71% of the substance was biodegraded. The pass level of 60% biodegradation was reached in a period of approximately 13 days immediately following the attainment of 10% biodegradation, so the 14-day window criterion was met. Based on these findings, the test substance is classified as readily biodegradable.

Description of key information

The biodegradation potential of the substance in water was determined in a screening study according to OECD TG 301D (Closed Bottle Test) and in compliance with GLP criteria. In this study 2.0 mg/L test substance was inoculated with secondary activated sludge of a city sewage treatment plant for 28 days under aerobic conditions in the dark. After the 28-day incubation period, the cumulative percentage biodegradation was calculated from the oxygen depletion (BOD/ThOD ratio). After the 28-day incubation period 71% of the substance was biodegraded.The pass level of 60% biodegradation was reached in a period of approximately 13 days immediately following the attainment of 10% biodegradation, sothe 14-day window criterion was met. Based on these findings, the test substance is classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information