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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:
04 August to 09 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification and river water instead of an effluent/extract/mixture was used as inoculum.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 12-14 August 2014 / signed on 6 October 2014
Specific details on test material used for the study:
No additional information
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Remarks:
river water without particles was used as inoculum
Details on inoculum:
- Source of inoculum: River water was sampled from the Rhine near Heveadorp, The Netherlands (03-08-2016). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
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 CONDITIONS
- Composition of medium: The river water used in the Closed Bottle test was spiked per liter of water with 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 not added to the river water to prevent nitrification.
- Test temperature: 22-24 °C
- Continuous darkness: Yes
- Deionized water containing no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification system.

TEST SYSTEM
- Culturing apparatus: Test was performed in 0.3 L Biological oxygen demand (BOD) bottles with glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate
- Method used to create aerobic conditions: No data
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a Eutech Cyberscan pH11 pH meter (Eutech Instruments, Nijkerk, The Netherlands). The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels: Yes
- Test performed in open system: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

OTHERS:
- 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).
- The biological oxygen demand (BOD) mg/mg of the test item and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test item and sodium acetate in the closed bottle, respectively. 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
Preliminary study:
None
Key result
Parameter:
% degradation (O2 consumption)
Value:
18
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
23
Sampling time:
60 d
Details on results:
- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the test item is 2.8 mg/mg.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test item in the Closed Bottle test was not determined, because possible toxicity of the test item obtained from the gum by ethanol extraction to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test item at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the initial test item concentration is expected.
- Test conditions: The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 8.0 (both controls and test). Temperatures were within the prescribed temperature range of 22 to 24°C.
- Biodegradability: Test item biodegraded by 18% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this test substance was biodegraded by 23% 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 and could be degraded further in time because a plateau of biodegradation has not been reached at the end of the test. The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Key result
Parameter:
ThOD
Value:
2.8 other: mg O2/mg
Results with reference substance:
The ThOD of sodium acetate is 0.8 mg/mg. Sodium acetate was degraded by 83% of its theoretical oxygen demand after 14 days.

Table 5.2.1/1: 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

8.0

8.2

4.3

8.1

7.9

8.0

4.2

Mean (M)

8.2

8.0

8.1

4.3

14

7.7

7.3

7.7

3.2

7.6

7.3

7.6

3.1

Mean (M)

7.7

7.3

7.7

3.2

21

7.4

6.7

7.5

 

7.3

6.5

7.4

 

Mean (M)

7.4

6.6

7.5

 

28

7.3

6.4

7.4

 

7.3

6.1

7.4

 

Mean (M)

7.3

6.3

7.4

 

42

7.0

5.9

 

 

7.0

5.7

 

 

Mean (M)

7.0

5.8

 

 

60

6.9

5.4

 

 

6.7

5.5

 

 

Mean (M)

6.8

5.5

 

 

 Ocs River water with nutrients and silica gel (DCM added and evaporated).

Ot River water with nutrients, test material (2.0 mg/L) and silica gel.

Oc River water with nutrients.

Oa River water with nutrients and sodium acetate (6.7 mg/L). 

Table 5.2.1/2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, 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

0.2

3.8

4

70

14

0.4

4.5

7

83

21

0.8

14

28

1.0

18

42

1.2

 

21

 

60

1.3

 

23

 

 

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.3 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 83. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Test item is biodegraded by 18% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this test substance was biodegraded by 23% at day 60. The test item should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrating that this substance is partially and inherently biodegradable and could be degraded further in time because a plateau of biodegradation has not been reached at the end of the test. A degradation >20% indicates that the substance is inherently degradable and that no parent substance will remain after 60 days. As the study was performed on river water the results are salient for environmental fate.
The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Executive summary:

A ready biodegradability test was performed according to the OECD Guideline 301 D, to determine the biodegradability of the test item by the Closed Bottle Test.

The test item was exposed to river water at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42 and 60 days. The degradation of the test item was assessed by the measurement of oxygen consumption. 

 

The test is valid as shown by an endogenous respiration of 1.3 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 83% 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.

 

The calculated theoretical oxygen demand (ThOD) of the test item is 2.8 mg/mg. Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test item in the Closed Bottle test was not determined, because possible toxicity of the test item obtained from the gum by ethanol extraction to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test item at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the initial test item concentration is expected.

 

Test item is biodegraded by 18% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this test substance was biodegraded by 23% at day 60. The test item should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrating that this substance is partially and inherently biodegradable and could be degraded further in time because a plateau of biodegradation has not been reached at the end of the test. A degradation >20% indicates that the substance is inherently degradable and that no parent substance will remain after 60 days. As the study was performed on river water the results are salient for environmental fate.

The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Description of key information

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

18% and 23% biodegradation after 28 and 60 days, respectively.

Not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

To assess the biodegradation potential of the registered substance, one experimental key study is available.

This valid study (AkzoNobel, 2016) was performed on the registered substance according to OECD Guideline No. 301D with GLP compliance. The substance was exposed to river water at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42 and 60 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. According to the results of this study, no inhibition of the biodegradation due to the initial test substance concentration is expected. The test substance was biodegraded by 18% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, this test substance was biodegraded by 23% at day 60. The substance should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrating that this substance is partially and inherently biodegradable and could be degraded further in time because a plateau of biodegradation has not been reached at the end of the test. A degradation >20% indicates that the substance is inherently degradable and that no parent substance will remain after 60 days. As the study was performed on river water, the results are salient for environmental fate. The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.