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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 September to 30 November 2015
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 (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:
No additional information
Oxygen conditions:
aerobic
Inoculum or test system:
other: 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 (24-09-2015).
- 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):
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 17025 certified; non-GLP analysis) was prepared in a water purification system.

TEST SYSTEM
- Culturing apparatus: Test was performed in 0.30 L BOD (biological oxygen demand) 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

SAMPLING
- Sampling frequency: 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, 28, 42 and 60.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: No

OTHERS:
- 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 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:
2
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
60 d
Remarks on result:
other: not readily biodegradable
Details on results:
- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the test substance is 3.0 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 (control test) and 8.0 (control with silica gel). Temperatures were within the prescribed temperature range of 22 to 24 °C.
- Biodegradability: Test substance was biodegraded by 2% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test (enhanced biodegradability test) the biodegradation of the test substance remained 2% at day 60. Test substance was not biodegraded in the Closed Bottle test and should therefore not be classified as readily biodegradable. Lack of biodegradation does not mean that test substance is persistent in nature. The stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Key result
Parameter:
ThOD
Value:
3 other: mg O2/mg
Results with reference substance:
The ThOD of sodium acetate is 0.8 mg/mg. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 89.

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

8.1

8.2

8.1

3.9

8.1

8.1

8.2

3.9

Mean (M)

8.1

8.2

8.2

3.9

14

7.8

7.9

7.9

3.0

7.9

7.8

7.9

3.1

Mean (M)

7.9

7.9

7.9

3.1

21

7.8

7.8

7.7

 

7.8

7.8

7.8

 

Mean (M)

7.8

7.8

7.8

 

28

7.7

7.4

7.5

 

7.6

7.6

7.7

 

Mean (M)

7.7

7.5

7.6

 

42

7.3

7.3

 

 

7.3

7.1

 

 

Mean (M)

7.3

7.2

 

 

60

6.9

7.0

 

 

7.0

6.7

 

 

Mean (M)

7.0

6.9

 

 

  

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

-0.1

4.3

0

80

14

0.0

4.8

0

89

21

0.0

 

0

 

28

0.1

 

2

 

42

0.2

 

3

 

60

0.1

 

2

 

 

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.1 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:
not readily biodegradable
Conclusions:
The test substance was biodegraded by 2% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the biodegradation of the test substance remained 2% at day 60 (enhanced biodegradability testing). This result demonstrates that the test substance should not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is persistent in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
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, 28, 42 and 60 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. The validity of the test is demonstrated by an endogenous respiration of 1.1 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.

 The calculated theoretical oxygen demand (ThOD) of the test substance is 3.0 mg/mg. 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 2% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the biodegradation of the test item remained 2% at day 60 (enhanced biodegradability testing). This result demonstrates that the test substance should not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is persistent in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non-GLP screening tests
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 (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:
no
Specific details on test material used for the study:
No additional information
Oxygen conditions:
aerobic
Inoculum or test system:
other: River water and activated sludge
Details on inoculum:
The Closed Bottle tests carried out were inoculated with either river water or activated sludge:
- Secondary activated sludge 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, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2 mg DW/L in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
- River water was sampled from the Rhine near Heveadorp, The Netherlands. This river water was aerated for 7 days before use and particles were removed by sedimentation. The river
water spiked with mineral salts was used undiluted.
Duration of test (contact time):
196 d
Initial conc.:
>= 0.5 - <= 2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 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, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Test temperature: 22 to 24°C
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 3 bottles containing only inoculum and 3 bottles with test substance and the respective inoculum.
- Test performed in closed vessels: yes
- Test performed in open system: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: No

OTHERS:
The test substance was administrated with the use of silica gel. 60 mg of test substance was dosed on 20 g of silica gel in a 50-mL serum flask. Only the upper layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw cap and mixed vigorously. The content of the bottle was seperated from the screw cap by a piece of alumiminium foil. Subsequently, 200 mg of silica gel was administrated to each test bottle. The final concentration of the test substance in the BOD bottles was 2.0 mg/L. Each of the prepared solutions were dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). The BOD mg/mg of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The biodegradation was calculated as the ratio of the BOD to the theoretical oxygen demand (ThOD).

The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530). The pH was measured using a EUTECH instruments pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger.


Preliminary study:
None
Key result
Parameter:
% degradation (O2 consumption)
Value:
>= -2 - <= 1
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
>= 43 - <= 47
Sampling time:
196 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
168 d
Remarks on result:
other: with activated sludge and lowest test substance concentration (1 mg/L) and pH set at 6.0, the substance would ultimately biodegrade after a long period of time
Details on results:
Inhibition was detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of a test substance especially with river water as inoculum. The test substance is therefore considered as toxic to the microorganisms present in the inocula. Decrease of the initial test substance concentration resulted in improved biodegradation. The pH of the media in the standard tests was 7.3±0.1 (activated sludge) and 8.0±0.1 (river water) at the start. The pH at day 28 was 7.2±0.1 (activated sludge) and 7.9±0.1 (river water). The pH of the media was lowered to increase the rate of chemical hydrolysis of the epoxide group (upon request). The pH in these two tests with river water ranged from 5.9 to 6.1. Temperatures ranged from 22 to 24°C. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The ThOD used to calculate the biodegradability percentages was 3.0 g O2/g test substance. No biodegradation of the test substance was found at day
28 in all screening tests carried out. Ready biodegradation of the test substance can therefore most likely not be demonstrated in the Closed Bottle test. After prolonged test periods (84 to 106 days) biodegradation in excess of 40% was found. Highest biodegradation percentages were obtained with the lowest test substance concentration (1.0 mg/L). The results demonstrate that the substance degrades at higher rates at lower pH. The prolonged Closed Bottle tests do demonstrate that most of the components of the test substance are susceptible to biodegradation. The biodegradation results obtained after 60 days do not allow to conclude that the substance is not persistent in the environment (applying Annex XIII REACH Criteria). However, in one screening test with activated sludge and lowest test substance concentration 1.0 mg/L, >60% biodegradation was achieved. Therefore the substance would ultimately biodegrade after a long period of time (biodegradation > 60% after 168 d), even though the substance meets the persistence criterion (applying Annex XIII REACH Criteria) and is not readily biodegradable (biodegradation <60% after 28d).
Parameter:
ThOD
Value:
3 g O2/g test mat.

Table 5.2.1/1: Results of the non-GLP screening test

Inoculum (initial concentration, mg/L)

Biodegradation percentage at day

7

14

21

28

42

56

84

112

140

196

River water (2.0)

-2

-1

-1

-1

1

-1

-2

-3

 

 

River water (1.0)

-2

-5

-2

-1

-3

1

9

22

34

43

River water (0.5)

-7

-7

-2

-2

-9

-2

7

16

31

43

River water (1.0)a

-1

2

2

1

7

18

41

49

57

 

River water (1.0)b

0

-2

-1

1

7

7

24

32

45

 

Sludge (2.0)

-1

0

1

-1

1

1

4

12

 

 

Sludge (1.0)

-1

-2

-2

1

1

2

10

26

40

47

Sludge (1.0)a

-1

2

2

1

7

18

41

49

57

62c

Sludge (1.0)b

0

-2

-1

1

7

7

24

32

45

 

a) pH set at 6.0

b) pH set at 6.0 and activated sludge concentration increased to 4.0 mg DW/L instead of 2.0 mg DW/L

c) day 168 instead of 196

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
No biodegradation of test substance was found at day 28 in all screening tests carried out. After prolonged test periods (84 to 106 days) biodegradation in excess of 40% was found. Highest biodegradation percentages were obtained with the lowest test substance concentration (1.0 mg/L). The results demonstrate that test substance degrades at higher rates at lower pH. The biodegradation results obtained after 60 days do not allow to conclude that test substance is not persistent in the environment (applying Annex XIII REACH Criteria). However, in one screening test with activated sludge and lowest test substance concentration 1.0 mg/L, >60% biodegradation was achieved. Therefore test substance would ultimately biodegrade after a long period of time (biodegradation > 60% after 168 d), even though test substance meets the persistence criterion (applying Annex XIII REACH Criteria) and is not readily biodegradable (biodegradation <60% after 28 days).
Executive summary:

In a non-GLP screening test, the test substance (at different concentrations) was exposed to river water and secondary activated sludge 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, 56, 84, 112, 140 and 196 days.The tests were performed in 0.3 L BOD bottles with glass stoppers; 3 bottles containing only inoculum and 3 bottles with test substance and the respective inoculum were used. The degradation of the test substance was assessed by the measurement of oxygen consumption. The BOD mg/mg of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The biodegradation was calculated as the ratio of the BOD to the theoretical oxygen demand (ThOD).

 

Inhibition was detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of a test substance especially with river water as inoculum. The test substance is therefore considered as toxic to the microorganisms present in the inocula. Decrease of the initial test substance concentration resulted in improved biodegradation. The pH of the media in the standard tests was 7.3±0.1 (activated sludge) and 8.0±0.1 (river water) at the start. The pH at day 28 was 7.2±0.1 (activated sludge) and 7.9±0.1 (river water). The pH of the media was lowered to increase the rate of chemical hydrolysis of the epoxide group. The pH in these two tests with river water ranged from 5.9 to 6.1. Temperatures ranged from 22 to 24°C. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

 

The ThOD used to calculate the biodegradability percentages was 3.0 g O2/g test substance. No biodegradation of test substance was found at day 28 in all screening tests carried out. Ready biodegradation of test substance can therefore most likely not be demonstrated in the Closed Bottle test. After prolonged test periods (84 to 106 days) biodegradation in excess of 40% was found. Highest biodegradation percentages were obtained with the lowest test substance concentration (1.0 mg/L). The results demonstrate that test substance degrades at higher rates at lower pH. The prolonged Closed Bottle tests do demonstrate that most of the components of test substance are susceptible to biodegradation. The biodegradation results obtained after 60 days do not allow to conclude that test substance is not persistent in the environment (applying Annex XIII REACH Criteria). However, in one screening test with activated sludge and lowest test substance concentration 1.0 mg/L, >60% biodegradation was achieved. Therefore test substance would ultimately biodegrade after a long period of time (biodegradation > 60% after 168 d), even though test substance meets the persistence criterion (applying Annex XIII REACH Criteria) and is not readily biodegradable (biodegradation <60% after 28 days).

Description of key information

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

2% biodegradation after 28 and 60 days (river water)

Non-inhibitory to the microorganisms at the tested concentration (2 mg/L)

Not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One experimental valid key study is available to assess the biodegradation potential of the registered substance, by the Closed Bottle Test, according to the OECD Guideline 301D and GLP compliance.

In this study, 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, 28, 42 and 60 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. The test substance did not cause a reduction in the endogenous respiration and therefore is considered to be non-inhibitory to the inoculum.The test substance was biodegraded by 2% at day 28, and, in the prolonged test, the biodegradation of the test item remained 2% at day 60 (enhanced biodegradability testing). This result demonstrates that the registered substance should not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is persistent in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

In addition to this GLP test, a non-GLP screening test was performed with this substance. The results demonstrate that test substance degrades at higher rates at lower pH. The prolonged Closed Bottle tests do demonstrate that most of the components of test substance are susceptible to biodegradation. The biodegradation results obtained after 60 days do not allow to conclude that test substance is not persistent in the environment (applying Annex XIII REACH Criteria). However, in one screening test with activated sludge and lowest test substance concentration 1.0 mg/L, >60% biodegradation was achieved. Therefore test substance would ultimately biodegrade after a long period of time (biodegradation > 60% after 168 d), even though test substance meets the persistence criterion (applying Annex XIII REACH Criteria) and is not readily biodegradable (biodegradation <60% after 28 days).