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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:
2015-09-16 to 2015-10-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 301 D with GLP statement. 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:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
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)
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:
- Storage condition of test material: Stored at ambient temperature in the dark
- Stability under storage conditions: Stable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum: River water was sampled from the Rhine near Heveadorp, The Netherlands (10-09-2015).
- The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. 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):
28 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, 33.3 mg Na2HPO4•2H2O, 22.5 mg MgSO4•7H2O, 27.5 mg CaCl2, and 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 copper was prepared in a water purification system.

TEST SYSTEM
- Culturing apparatus: 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
- Toxicity control: No

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.
- The biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance 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:
71
Sampling time:
28 d
Remarks on result:
other: readily biodegradable based on the >60% biodegradation at day 28
Remarks:
10 day window not fulfilled
Details on results:
- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the test substance is 2.1 mg/mg.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance tested was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound 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 7.9 (test) and 8.0 (controls). Temperatures were within the prescribed temperature range of 22-24 °C.
- Biodegradability: ST11 C13 was biodegraded by 71% at day 28 in the Closed Bottle test. ST11 C13 is classified as readily biodegradable based on the >60% biodegradation at day 28. The test substance did not fulfill the time window criterion for ready biodegradable substance. ST11 C13 consists of 2-propanol, lactate, and isobutyric acid linked together with ester bonds. Biodegradation is usually initiated by hydrolysis of the ester bonds. Biodegradation of the hydrolysis products requires the concerted action of different microorganisms as a single organism usually lacks the full complement of enzymatic capabilities. The biodegradation of 2-propanol, lactate, and isobutyric acid may be fully in line with the time window criterion when judged as separate chemicals. The biodegradation of the hydrolysis products by different microorganisms is the reason for not applying the time window as pass/fail criterion.
Key result
Parameter:
ThOD
Value:
2.1 other: mg O2/mg test material
Results with reference substance:
The ThOD of sodium acetate is 0.8 mg/mg. Sodium acetate was degraded by 89% 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

8.0

7.8

7.9

3.5

7.9

7.8

8.0

3.9

Mean (M)

8.0

7.8

8.0

3.7

14

7.7

5.5

7.6

2.7

7.7

5.8

7.6

2.9

Mean (M)

7.7

5.7

7.6

2.8

21

7.4

5.0

7.4

-

7.4

5.2

7.6

-

Mean (M)

7.4

5.1

7.5

-

28

7.3

4.2

7.3

-

7.2

4.4

7.3

-

Mean (M)

7.3

4.3

7.3

-

 

Ocs River water with nutrients and silica gel.

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

4.3

5

80

14

2.0

4.8

48

89

21

2.3

-

55

-

28

3.0

-

71

-

 

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.4 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 89. Finally, the validity of the test is shown with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test substance was biodegraded by 71% at day 28 in the Closed Bottle test. ST11 C13 is classified as readily biodegradable based on the >60% biodegradation at day 28. Hence, this substance should be classified as readily biodegradable. The test substance did not fulfill the time window criterion for ready biodegradable substance.
Executive summary:

This study was performed according to the OECD Guideline 301 D, to determine the biodegradability of the test substance by the Closed Bottle Test.

The test 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 and 28 days. The degradation of the test material was assessed by the measurement of oxygen consumption. 

The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 89% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

 

The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. ST11 C13 was biodegraded by 71% at day 28 in the Closed Bottle test. ST11 C13 is classified as readily biodegradable based on the >60% biodegradation at day 28. The test substance did not fulfill the time window criterion for ready biodegradable substance. ST11 C13 consists of 2-propanol, lactate, and isobutyric acid linked together with ester bonds. Biodegradation is usually initiated by hydrolysis of the ester bonds. Biodegradation of the hydrolysis products requires the concerted action of different microorganisms as a single organism usually lacks the full complement of enzymatic capabilities. The biodegradation of 2-propanol, lactate, and isobutyric acid may be fully in line with the time window criterion when judged as separate chemicals. The biodegradation of the hydrolysis products by different microorganisms is the reason for not applying the time window as pass/fail criterion.

 

The test substance was biodegraded by 71% at day 28 in the Closed Bottle test. ST11 C13 is classified as readily biodegradable based on the >60% biodegradation at day 28. Hence, this substance should be classified as readily biodegradable. The test substance did not fulfill the time window criterion for ready biodegradable substance.

Description of key information

OECD Guideline 301D, GLP, key study, validity 2:

71% biodegradation after 28 days (inoculum: river water). Readily biodegradable but failing 10-day window.

Even if the 10-day window criterion for ready biodegradable substance is not fulfilled, as >70% biodegradation were observed after 28 days under environmentally realistic conditions (river water), the criterion of rapid degradability is met, according to CLP Regulation No 1272/2008.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

One valid key study is available (AkzoNobel, 2015) to assess the readily biodegradation of the registered substance.

This study was performed on the registered substance according to the OECD Guideline 301 D and GLP. The test 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 and 28 days. The degradation of the test material was assessed by the measurement of oxygen consumption. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 89% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period. The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum.

The test substance was biodegraded by 71% at day 28 in the Closed Bottle test but did not fulfill the 10-day window criterion for ready biodegradable substance. However, the substance biodegradability criterion of >60% biodegradation at day 28 was met. The test material consists of 2-propanol, lactate, and isobutyric acid linked together with ester bonds. Biodegradation is usually initiated by hydrolysis of the ester bonds. Biodegradation of the hydrolysis products requires the concerted action of different microorganisms as a single organism usually lacks the full complement of enzymatic capabilities. The biodegradation of 2-propanol, lactate, and isobutyric acid may be fully in line with the time window criterion when judged as separate chemicals. The biodegradation of the hydrolysis products by different microorganisms can be the reason for not applying the time window as pass/fail criterion. However, even if the 10-day window criterion for ready biodegradable substance is not fulfilled, as >70% biodegradation were observed after 28 days under environmentally realistic conditions (river water), the criterion of rapid degradability is met, according to CLP Regulation No 1272/2008.