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Environmental fate & pathways

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:
18 September 2013 - 20 November 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Read-across to study performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
(Prolongation up to 60 days, ammonium chloride omitted from medium to prevent oxygen consumption due to nitrification (no nitrogen limitation as shown by biodegradation of reference compound), inoculum activated sludge instead of effluent/surface water)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Secondary activated sludge (12-09-13) from the STP Nieuwgraaf in Duiven, The Netherlands - treating predominatntly domestic wastewater.
- Storage conditions: activated sludge was preconditioned to reduce the endogenous respiration rates: 400 mg dry Weight (DW)/L of activated sludge was aerated for one week. Sludge was diluted in the biological oxygen demand (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 CONDITIONS
- Composition of medium:
Nutrients 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 to prevent nitrification.

- Solubilising agent: Dichloromethane (Sigma-Aldrich)
Sodium acetate was added to the bottles using an aqueous stock solution of 1.0 g/L. Linseed standoil was added to the bottles using a stock of 1.0 g/L in DCM.
For the Closed Bottle test, the test substance in DCM (0.6 mL) was directly added to the bottles. The bottles were placed on a roller bank in a ventilated hood for a few hours to allow the solvent to evaporate and to obtain an even distribution of the test substance on the walls of the bottles.
- Test temperature: 22°C -24°C
- pH: at day 0: 7.2
at day 28: 7.3 (both controls) and 7.2 (test)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles containing only inoculum; 6 bottles containing inoculum and sodium acetate; 10 bottles containing inoculum and test substance; and 10 bottles treated with DCM containing inoculum.
- Measuring equipment: electronical measurement of the oxygen concentration using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands); pH was measured using a Eutech Cyberscan pH11 pH meter (Eutech Instruments, Nijkerk, The Netherlands)

SAMPLING
- Sampling frequency: day 0, 7, 14, 21 and 28
- Sampling method: two duplicate bottles of all series were withdrawn for dissolved oxygen analyses on the respective days; One extenstion of the Closed bottle test by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. An oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. Medium dissipated by the electrode flowed back into the BOD bottle after withdrawal of the oxgen electrode from the funnel (van ginkel and Stroo 1992). This method allowed measurement at day 42 and 60.

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculum alone and inoculum with DCM
Key result
Parameter:
% degradation (O2 consumption)
Value:
33
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
41
Sampling time:
60 d
Remarks on result:
other: prolonged Closed Bottle test
Details on results:
See table under 'Any other information'
The calculated theoretical oxygen demand (ThOD) of linseed standoil is 2.9 mg/mg and the ThOD of sodium acetate is 0.8 mg/mg.
The inhibition of the degradation of sodium acetate by the test substance in the Closed Bottle test was not determined due to irrelevent possible toxicity of linseed standoil to microorganisms degrading acetate. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Thus, no inhibition of the biodegradation expected.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 81.

Oxygen consumption (mg/L) and biodegradation (%) of linseed standoil (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

0.9

4.1

19

76

14

1.3

4.4

22

81

21

1.7

 

29

 

28

1.9

 

33

 

42

2.0

 

34

 

60

2.4

 

41

 

Validity criteria fulfilled:
yes
Remarks:
Difference of the replicate values at day 28 < 20%; Biodegradation percentage of the reference compound (sodium acetate) was 81 at day 14; Oxygen concentration > 0.5 mg/L in all bottles during the test period.
Interpretation of results:
inherently biodegradable
Conclusions:
Linseed standoil is biodegraded by 33% at day 28 and 41% at day 60 (prolonged Closed Bottle test). Linseed standoil is therefore not readily biodegradable, but can be classified as inherently biodegradable.
Executive summary:

A ready biodegradability test in an aerobic aqueous medium was conducted to assess the biotic degradation of linseed standoil. The used Closed Bottle test was performed according to slightly modified OECD, EU and ISO Test Guidelines and in compliance with the OECD principles of Good Laboratory Practice.

Linseed standoil is considered non-inhibitory to the inoculum as it did not cause a reduction in the endogenous respiration. The Linseed stand was degraded by 33% on day 28 and 41% on day 60 in the Closed Bottle test. Thus the test substances revealed to not be readily biodegradable, but can be classified as inherently biodegradable.

The test fullfils the validity criteria by an endogenous respiration of 0.9 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 81% of its theoretical oxygen demand after 14 days. Also, all bottles contained an oxygen concentration of >0.5 mg/L during the entire test period.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Key result
Parameter:
% degradation (O2 consumption)
Value:
33
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
41
Sampling time:
60 d
Remarks on result:
other: prolonged Closed Bottle test
Details on results:
See table under 'Any other information'
The calculated theoretical oxygen demand (ThOD) of linseed standoil is 2.9 mg/mg and the ThOD of sodium acetate is 0.8 mg/mg.
The inhibition of the degradation of sodium acetate by the test substance in the Closed Bottle test was not determined due to irrelevent possible toxicity of linseed standoil to microorganisms degrading acetate. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Thus, no inhibition of the biodegradation expected.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 81.

Oxygen consumption (mg/L) and biodegradation (%) of linseed standoil (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

0.9

4.1

19

76

14

1.3

4.4

22

81

21

1.7

 

29

 

28

1.9

 

33

 

42

2.0

 

34

 

60

2.4

 

41

 

Validity criteria fulfilled:
yes
Remarks:
Difference of the replicate values at day 28 < 20%; Biodegradation percentage of the reference compound (sodium acetate) was 81 at day 14; Oxygen concentration > 0.5 mg/L in all bottles during the test period.
Interpretation of results:
inherently biodegradable
Conclusions:
Linseed standoil is biodegraded by 33% at day 28 and 41% at day 60 (prolonged Closed Bottle test). Linseed standoil is therefore not readily biodegradable, but can be classified as inherently biodegradable.
Executive summary:

A ready biodegradability test in an aerobic aqueous medium was conducted to assess the biotic degradation of linseed standoil. The used Closed Bottle test was performed according to slightly modified OECD, EU and ISO Test Guidelines and in compliance with the OECD principles of Good Laboratory Practice.

Linseed standoil is considered non-inhibitory to the inoculum as it did not cause a reduction in the endogenous respiration. The Linseed stand was degraded by 33% on day 28 and 41% on day 60 in the Closed Bottle test. Thus the test substances revealed to not be readily biodegradable, but can be classified as inherently biodegradable.

The test fullfils the validity criteria by an endogenous respiration of 0.9 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 81% of its theoretical oxygen demand after 14 days. Also, all bottles contained an oxygen concentration of >0.5 mg/L during the entire test period.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 January 2010 - 25 February 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to study performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Source:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment:
The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (73 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
31 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: Total Organic Carbon (TOC)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

- Test temperature: between 21.7 and 22.2°C.
- pH:
At t=0 d: 7.5 - 7.6
At t=28 d: 7.7 - 7.9
- pH adjusted:no
- Aeration of dilution water: Not before the test, the test is aerated continously
- Suspended solids concentration: The concentration of suspended solids was 3.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%)) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used:
CO2 was trapped in barium hydroxide solution. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.


SAMPLING
- Sampling frequency: Titration were made on day: 2, 5, 7, 9, 14, 19, 23, 27 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other:
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
13
Sampling time:
29 d
Remarks on result:
other: HCL added on the 28th day (last CO2-measurement on the 29th day)
Parameter:
% degradation (CO2 evolution)
Value:
16
Sampling time:
29 d
Remarks on result:
other: HCL added on the 28th day (last CO2-measurement on the 29Th day)
Details on results:
In the toxicity control more than 25% biodegradation occurred within 14 days (41%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The positive control substance was biodegraded by at least 60% (77%) within 14 days.
Validity criteria fulfilled:
yes
Interpretation of results:
other: The relative biodegradation values revealed 13 and 16% biodegradation of Standolized linseed oil, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
Conclusions:
Standolized linseed oil was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Key result
Parameter:
% degradation (CO2 evolution)
Value:
13
Sampling time:
29 d
Remarks on result:
other: HCL added on the 28th day (last CO2-measurement on the 29th day)
Parameter:
% degradation (CO2 evolution)
Value:
16
Sampling time:
29 d
Remarks on result:
other: HCL added on the 28th day (last CO2-measurement on the 29Th day)
Details on results:
In the toxicity control more than 25% biodegradation occurred within 14 days (41%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The positive control substance was biodegraded by at least 60% (77%) within 14 days.
Validity criteria fulfilled:
yes
Interpretation of results:
other: The relative biodegradation values revealed 13 and 16% biodegradation of Standolized linseed oil, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
Conclusions:
Standolized linseed oil was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
February 10 - March 31, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Read-across to study not according to a recommended OECD 301 or 302 guideline, and with reporting deficiencies. GLP compliant.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Method for the determination of ecotoxicity at level 1, Biodegradation; Repetitive Die Away Test, DG XI/400/84, Rev. 1, EEC (1984)
Deviations:
yes
Remarks:
Test medium does not contain NH4Cl and the acclimation of the sludge takes place in a 1 liter vessel at 350 mg SS/L
Principles of method if other than guideline:
The test medium is inoculated with activated sludge, stabilized for one week and then spiked with the test substance. After two weeks, three repetitive weekly additions of test substance take place. After each repetitive addition, the water phase is re-aerated to the saturation level. The oxygen consumption is measured weekly up to two weeks after the last addition. The percentage biodegradation is calculated from the measured oxygen consumption and the analytically determined Chemical Oxygen demand (COD).
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal waste water treatment plant of Duiven, The Netherlands, receiving only waste water from domestic origin
- Storage length: preconditioned during a week (unspecified conditions)
- Concentration of sludge: final concentration in test flasks 35 mg suspended solids/L.
Duration of test (contact time):
14 d
Initial conc.:
40 mg/L
Based on:
test mat.
Initial conc.:
ca. 120 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test medium was inoculated with activated sludge (35 mg suspended solids/mL), stabilized for one week and then spiked with the test substance (40 mg/L). The following emulsifiers were used: genapol PF-40, 20% of the weight of the oil; Nonylphenol.10EO.5PO, 40% of the weight of the oil. It was reported that these emulsifiers are not toxic in the biodegradation test and not biodegradable. Control solutions contained inoculums and emulsifier but no test substance. Triplicate flasks (dark glass bottles containing 187 mL of liquid and 93 L of gas space) were used for the treated and the control test solutions. The flasks were incubated at 20°C and were shaken continuously to assure steady state oxygen partitioning between the liquid and the gas phase. After two weeks, three repetitive weekly additions of test substance were performed. After each repetitive addition, the water phase was re-aerated to the saturation level. The oxygen consumption was measured weekly up to two weeks after the last addition. The percentage biodegradation was calculated from the measured oxygen consumption in the treated test solutions, corrected for the oxygen consumption in the control solutions, and the analytically determined Chemical Oxygen Demand (2.36 g/g) of the test substance. A toxicity control was included (sodium acetate in the presence of 7.6 mg oil/L).
Reference substance:
acetic acid, sodium salt
Remarks:
(concentration not reported)
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
20
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
21 d
Remarks on result:
other: total % biodegradation for first two additions one week after second addition
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
28 d
Remarks on result:
other: total % biodegradation for first three additions one week after third addition
Key result
Parameter:
% degradation (O2 consumption)
Value:
19
Sampling time:
42 d
Remarks on result:
other: total % biodegradation for four additions two weeks after fourth addition
Details on results:
The biodegradation of sodium acetate is not inhibited in the presence of 7.6 mg/L
Results with reference substance:
About 75% biodegradation within one week (read from the graph)

Results:

parameter

day 0

day 7

day 14

day 21

day 28

day 35

day 42

total oxygen uptake (mg)

-

2.5

3.6

5.4

9.7

12.8

16.0

total oxygen demand (mg)

17.9

17.9

17.9

38.9

68.1

84.8

84.8

% biodegradation

-

14

20

14

14

15

19

 

Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
In a test measuring oxygen uptake (not according to any OECD 301 or 302 guideline), 20% biodegradation was recorded after 14 days exposure to non-adapted inoculums, and 19% biodegradation two weeks after the last of three subsequent weekly additions to adapted inoculum. The rate of biodegradation may have been impaired by problems of solubility and mass transfer.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
20
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
21 d
Remarks on result:
other: total % biodegradation for first two additions one week after second addition
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
28 d
Remarks on result:
other: total % biodegradation for first three additions one week after third addition
Key result
Parameter:
% degradation (O2 consumption)
Value:
19
Sampling time:
42 d
Remarks on result:
other: total % biodegradation for four additions two weeks after fourth addition
Details on results:
The biodegradation of sodium acetate is not inhibited in the presence of 7.6 mg/L
Results with reference substance:
About 75% biodegradation within one week (read from the graph)

Results:

parameter

day 0

day 7

day 14

day 21

day 28

day 35

day 42

total oxygen uptake (mg)

-

2.5

3.6

5.4

9.7

12.8

16.0

total oxygen demand (mg)

17.9

17.9

17.9

38.9

68.1

84.8

84.8

% biodegradation

-

14

20

14

14

15

19

 

Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
In a test measuring oxygen uptake (not according to any OECD 301 or 302 guideline), 20% biodegradation was recorded after 14 days exposure to non-adapted inoculums, and 19% biodegradation two weeks after the last of three subsequent weekly additions to adapted inoculum. The rate of biodegradation may have been impaired by problems of solubility and mass transfer.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 March 2013 - 21 Feb 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Read-across to study performed according to OECD and/or EC guidelines, not under GLP ( preliminary study).
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Prolongation up to 112 days, only one series of test bottles was used to measure the oxygen concentration (so the same bottles were measured each week))
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge, domestic, non adapted OR Natural River water
Details on inoculum:
- Source of inoculum/activated sludge:
Closed Bottle tests were started with two different inocula:
1. Secondary activated sludge from the STP Nieuwgraaf in Duiven, The Netherlands - treating predominantly domestic wastewater.
The activated sludge was preconditioned to reduce the endogenous respiration rates: 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. Sludge was diluted in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992). The activated sludge was diluted in the mineral medium to 2 mg dw/L
2. River water was taken from the River Rhine near Heveadorp, The Netherlands. This river water was aerated for 7 days before use and particles were removed by sedimentation.
SCAS test: secondary activated sludge (approx. 2 g dw/L), taken from the same STP Nieuwgraaf.

- Storage conditions and duration:
Inocula for Closed Bottle test: activated sludge was preconditioned to reduce endogenous respiration rates: sludge at 400 mg dw/L was aerated for one week; River water was also aerated for one week

- Preparation of inoculum for exposure and concentration:
Closed bottle test: activated sludge was diluted to 2 mg dw/L in mineral medium
River water was used as such (with addition of minerals)
Inoculum for SCAS test: Started with approx 3 g dw/L
Duration of test (contact time):
>= 56 - <= 112 d
Initial conc.:
>= 1 - <= 2 mg/L
Based on:
test mat.
Initial conc.:
50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
in the Closed Bottle test
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
in the SCAS test
Details on study design:
TEST CONDITIONS
- Composition of medium:
Nutrients per liter of deionized or river 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 to prevent nitrification.

- Solubilising agent:
Genapol PF40 (Clariant Sulzbach, Germany)
Tween 80 and Tween 85 (Sigma Aldrich, Zwijndrecht, The Netherlands)
Synperonic PE/P105 (Uniqema Wilton, UK)
First a stock solution (1.0 g/L) was prepared of Stand Linseed oil in dichloromethane (reagent grade)
For the Closed Bottle test, the test substance in dichloromethane (0.3 and 0.6 mL) was directly added to the bottles. The bottles were placed on a roller bank in a ventilated hood for 4 hours to allow the solvent to evaporate and to obtain an even distribution of the test substance on the walls of the bottles. Stock dispersions of linseed standoil with Synperonic PE/P105, Genapol PF40, Tween 80, or Tween 85 were prepared by agitating volumes of approximately 1.0 L overnight with magnetic stirrer bars. The concentration of linseed standoil, and surfactants in the stock dispersions was 1.0 g/L. These disersions were added to the bottles. The concentrations of the test substance (and surfactant) in the bottles were 1.0 and 2.0 mg/L.
Each of the prepared solutions and dispersions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.
For the SCAS test, a stock dispersion with the test substance and Tween 80 both at a concentration of 1.0 g/L was used. Each day at the fill and draw procedure (100 ml), 5 ml of the test substance dispersion was added to the final volume of 150 ml (resulting in addition of 33.3 mg/L in the SCAS Unit)

- Test temperature: 23˚C
- pH: media with sludge: pH 7.0±0.2; river water: pH 8.0 ±0.1
- Suspended solids concentration: Closed Bottle test: 2 mg dw/L; SCAS test: 3 g dw/L
- Light: Closed bottle test: dark ; SCAS test: ambient light
- Other: SCAS test fed daily with primary settled sewage

TEST SYSTEM
- Culturing apparatus:
Closed Bottle test: 0.3 L-BOD bottles with glass stoppers; the bottles were filled, closed and incubated in the dark at 23˚C. One series of bottles containing magnetic bars was placed on stirrer plates (600 rpm).
SCAS test: 150 ml-SCAS Units
- Number of culture flasks/concentration: 3 bottles per treatment. Treatments include:
Inoculum / Inoculum with different surfactants / Inoculum with test substance (and different surfactants)
- Measuring equipment: oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (closed bottle test); TOC apparatus (Shimadzu) (SCAS test)

SAMPLING
- Sampling frequency:
Closed Bottle test: week 1, 2, 3, 4, 7, 8, 12, 16
SCAS test: 3 consecutive days after 1 month and after 10 weeks
- Sampling method:
SCAS test: Daily: aeration was stopped and sludge was allowed to settle for 45 minutes. After settling 100 mL of the supernatant liquor was withdrawn from the tap. Effluents were filtered using cellulose nitrate filters with pores of 8.0 µm to remove sludge particles (before determination of DOC).

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculum alone and inoculum with different surfactants
- Toxicity control: The absence of toxicity of the test substance is assessed against the endogenous respiration of the inoculum. If the endogenous respiration is in line with normally expected activity, inhibition is not expected.
Parameter:
% degradation (O2 consumption)
Value:
>= 34 - <= 36
Sampling time:
28 d
Remarks on result:
other: Closed Bottles: 2 mg test substance/L and DCM
Parameter:
% degradation (O2 consumption)
Value:
45
Sampling time:
56 d
Remarks on result:
other: Closed Bottles: 2 mg test substance/L and DCM
Parameter:
% degradation (DOC removal)
Value:
>= 72 - <= 74
Sampling time:
31 d
Remarks on result:
other: SCAS test: daily 50 mg test subst/L and Tween 80
Parameter:
% degradation (DOC removal)
Value:
58
Sampling time:
70 d
Remarks on result:
other: SCAS test: daily 50 mg test subst/L and Tween 80
Details on results:
See table under 'Any other information'
Inhibition of the endogenous respriation was not detected. The validity of the Closed Bottle test is demonstrated by oxygen concentrations > 0.5 mg/L in all bottles during the test period.

Closed Bottle tests:
The biodegradation starts immediately but already levels off after 2 weeks. The biodegradation percentages reached at the end of the tests ranged from 27 to 45. Methods (agitation, surfactants etc) to increase the bioavailability did not result in higher biodegradation percentages. The biodegradation found at the end of the Closed Bottle tests strongly indicates that linseed standoil is only partially biodegradable.

SCAS test:
The carbon removal in the SCAS unit ranging from 58 to 74% also suggests partial degradation.

Closed Bottle tests with previously exposed SCAS inocula:
The partial degradation of linseed standoil was further underpinned by determining the biodegradation of linseed standoil in Closed Bottle tests inoculated with sludge from the SCAS test unit. Biodegradation did not improve when activated sludge exposed to the test substance for 4 and 9 week was used.

Closed Bottle tests, degradation expressed as the percentage BOD/ThOD in time

Method (inoculum) <initial concentration in mg/L>

Biodegradation at day

7

14

21

28

42

56

84

112

Synperonic (river) <1.0>

14

17

18

18

24

29

29

 

Genapol (river) <1.0>

6

16

16

22

24

25

27

 

Tween 80 (river) <1.0>

11

23

27

27

35

38

39

 

Tween 85 (river) <1.0>

14

18

22

23

31

33

38

37

DCM (river) <2.0>

11

18

21

21

26

28

 

 

DCM (river) <1.0>

8

10

13

15

19

21

 

 

 

 

 

 

 

 

 

 

 

DCM (sludge) <2.0>

19

29

32

36

42

45

 

 

DCM (sludge) <2.0>

20

26

32

34

37

38

38

 

Tween 80 (sludge) <2.0>

13

20

22

23

23

27

 

 

Tween 80 (sludge) <2.0> agitated

14

20

23

27

26

29

 

 

 

 

 

 

 

 

 

 

 

DCM (sludge exposed 4 weeks) <2.0>

19

25

29

31

34

35

36

 

DCM (sludge exposed 9 weeks) <2.0>

8

16

19

26

30

33

 

 

SCAS test, Organic carbon concentration and removal

Control unit fed with sewage only; test unit fed with an emulsion of Linseed Oil and Tween 80. Carbon removal is based on comparison with the control.

Time (days )

NPOC (mg/L)

Carbon removal (%)

Control

Test

30

7.1

18.5

70

31

8.1

18.8

72

32

8.2

18.1

74

69

10.7

24.5

64

70

12.3

25.9

65

71

12.4

28.4

58

Validity criteria fulfilled:
yes
Remarks:
oxygen concentration remained > 0.5 mg/l
Interpretation of results:
other: partially biodegradable
Conclusions:
The biodegradation in the Prolonged Closed Bottle Test ranged from 21 to 45% mineralisation. Methods (agitation, surfactants etc) to increase the bioavailability did not result in higher biodegradation percentages. The biodegradation found at the end of the Closed Bottle tests strongly indicates that linseed standoil is only partially biodegradable. The partial degradation was further underpinned by the results of the Closed Bottle tests inoculated with sludge from the SCAS test unit. Biodegradation did not improve when activated sludge exposed to the test substance for 4 and 9 week was used.
The biodegradation percentage in the enhanced biodegradability test exceeds 20%. Linseed standoil most likely readily converts into water, CO2, persistent water soluble substances and biomass.
Executive summary:

A preliminary study was performed to assess the biodegradabililty of Stand Linseed Oil. Various options to optimise the bioavailability of the test substance in the Closed Bottle Test (OECD TG 301D) were assessed, including agitation, use of the solvent Dichloromethane and dispersants Tween 80, Tween 85, Synperonic and Genapol. Two different inocula were included: activated sludge and river water.

The results indicated a partial biodegradation in the closed bottle test, also after prolonged exposure (21 - 45% mineralisation).

A SCAS test was performed to test the ultimate biodegradability under more favorable conditions (OECD TG 302A). The removal ranged from 58 to 74%, also suggesting partial degradation. The biodegradation did not increase when activated sludge of the SCAS units (pre-exposed for 4 and 9 weeks) was used as inoculum in the Closed Bottle tests, so adaptation does not take place.

Hydrolysis of the ester bonds and subsequent degradation of glycerol and fatty acids would result in a 65% carbon removal of linseed standoil (based on the assumed structure). The persistent parts of linseed standoil are most likely the modified fatty acids and/or partially degraded modified fatty acids. The far-ends of the alkyl chains can be oxidized and carboxylated far-end might be shortened through β-oxidation. This would result in water soluble substances detected as carbon in the effluent of the SCAS unit.

The biodegradation percentage found in the enhanced biodegradability test (Closed Bottle test) exceeds 20%. Linseed standoil most likely readily converts into water, CO2, persistent water soluble substances and biomass.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Parameter:
% degradation (O2 consumption)
Value:
>= 34 - <= 36
Sampling time:
28 d
Remarks on result:
other: Closed Bottles: 2 mg test substance/L and DCM
Parameter:
% degradation (O2 consumption)
Value:
45
Sampling time:
56 d
Remarks on result:
other: Closed Bottles: 2 mg test substance/L and DCM
Parameter:
% degradation (DOC removal)
Value:
>= 72 - <= 74
Sampling time:
31 d
Remarks on result:
other: SCAS test: daily 50 mg test subst/L and Tween 80
Parameter:
% degradation (DOC removal)
Value:
58
Sampling time:
70 d
Remarks on result:
other: SCAS test: daily 50 mg test subst/L and Tween 80
Details on results:
See table under 'Any other information'
Inhibition of the endogenous respriation was not detected. The validity of the Closed Bottle test is demonstrated by oxygen concentrations > 0.5 mg/L in all bottles during the test period.

Closed Bottle tests:
The biodegradation starts immediately but already levels off after 2 weeks. The biodegradation percentages reached at the end of the tests ranged from 27 to 45. Methods (agitation, surfactants etc) to increase the bioavailability did not result in higher biodegradation percentages. The biodegradation found at the end of the Closed Bottle tests strongly indicates that linseed standoil is only partially biodegradable.

SCAS test:
The carbon removal in the SCAS unit ranging from 58 to 74% also suggests partial degradation.

Closed Bottle tests with previously exposed SCAS inocula:
The partial degradation of linseed standoil was further underpinned by determining the biodegradation of linseed standoil in Closed Bottle tests inoculated with sludge from the SCAS test unit. Biodegradation did not improve when activated sludge exposed to the test substance for 4 and 9 week was used.

Closed Bottle tests, degradation expressed as the percentage BOD/ThOD in time

Method (inoculum) <initial concentration in mg/L>

Biodegradation at day

7

14

21

28

42

56

84

112

Synperonic (river) <1.0>

14

17

18

18

24

29

29

 

Genapol (river) <1.0>

6

16

16

22

24

25

27

 

Tween 80 (river) <1.0>

11

23

27

27

35

38

39

 

Tween 85 (river) <1.0>

14

18

22

23

31

33

38

37

DCM (river) <2.0>

11

18

21

21

26

28

 

 

DCM (river) <1.0>

8

10

13

15

19

21

 

 

 

 

 

 

 

 

 

 

 

DCM (sludge) <2.0>

19

29

32

36

42

45

 

 

DCM (sludge) <2.0>

20

26

32

34

37

38

38

 

Tween 80 (sludge) <2.0>

13

20

22

23

23

27

 

 

Tween 80 (sludge) <2.0> agitated

14

20

23

27

26

29

 

 

 

 

 

 

 

 

 

 

 

DCM (sludge exposed 4 weeks) <2.0>

19

25

29

31

34

35

36

 

DCM (sludge exposed 9 weeks) <2.0>

8

16

19

26

30

33

 

 

SCAS test, Organic carbon concentration and removal

Control unit fed with sewage only; test unit fed with an emulsion of Linseed Oil and Tween 80. Carbon removal is based on comparison with the control.

Time (days )

NPOC (mg/L)

Carbon removal (%)

Control

Test

30

7.1

18.5

70

31

8.1

18.8

72

32

8.2

18.1

74

69

10.7

24.5

64

70

12.3

25.9

65

71

12.4

28.4

58

Validity criteria fulfilled:
yes
Remarks:
oxygen concentration remained > 0.5 mg/l
Interpretation of results:
other: partially biodegradable
Conclusions:
The biodegradation in the Prolonged Closed Bottle Test ranged from 21 to 45% mineralisation. Methods (agitation, surfactants etc) to increase the bioavailability did not result in higher biodegradation percentages. The biodegradation found at the end of the Closed Bottle tests strongly indicates that linseed standoil is only partially biodegradable. The partial degradation was further underpinned by the results of the Closed Bottle tests inoculated with sludge from the SCAS test unit. Biodegradation did not improve when activated sludge exposed to the test substance for 4 and 9 week was used.
The biodegradation percentage in the enhanced biodegradability test exceeds 20%. Linseed standoil most likely readily converts into water, CO2, persistent water soluble substances and biomass.
Executive summary:

A preliminary study was performed to assess the biodegradabililty of Stand Linseed Oil. Various options to optimise the bioavailability of the test substance in the Closed Bottle Test (OECD TG 301D) were assessed, including agitation, use of the solvent Dichloromethane and dispersants Tween 80, Tween 85, Synperonic and Genapol. Two different inocula were included: activated sludge and river water.

The results indicated a partial biodegradation in the closed bottle test, also after prolonged exposure (21 - 45% mineralisation).

A SCAS test was performed to test the ultimate biodegradability under more favorable conditions (OECD TG 302A). The removal ranged from 58 to 74%, also suggesting partial degradation. The biodegradation did not increase when activated sludge of the SCAS units (pre-exposed for 4 and 9 weeks) was used as inoculum in the Closed Bottle tests, so adaptation does not take place.

Hydrolysis of the ester bonds and subsequent degradation of glycerol and fatty acids would result in a 65% carbon removal of linseed standoil (based on the assumed structure). The persistent parts of linseed standoil are most likely the modified fatty acids and/or partially degraded modified fatty acids. The far-ends of the alkyl chains can be oxidized and carboxylated far-end might be shortened through β-oxidation. This would result in water soluble substances detected as carbon in the effluent of the SCAS unit.

The biodegradation percentage found in the enhanced biodegradability test (Closed Bottle test) exceeds 20%. Linseed standoil most likely readily converts into water, CO2, persistent water soluble substances and biomass.

Description of key information

Not readily biodegradable: 33% mineralisation (oxygen consumption) in 28 days (OECD TG 301D, GLP study, Rel. 1). Prolongation of the study showed 41% mineralisation after 60 days. The test substance is considered to be inherently biodegradable.  

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

The ready biodegradability of Standolized soybean oil was read-across from various tests performed with Standolized linseed oil, which are summarized below. Based on the results, it is concluded that Soybean oil, polymerized is not readily biodegradable; however, the test substance is inherently biodegradable, not fulfilling specific criteria.

The degradation of Linseed oil, polymerized was tested in different tests for screening ready biodegradability. A study was designed to the bioavailability of the test substance using agitation, a solvent or dispersants. The table summarises the efforts and the results of all studies.

 

Remarks

% Degradation

Referentie

OECD 301D

Closed Bottle test

Inoculum: activated sludge

 

 

28d: 33% of ThOD

60d: 41% of ThOD

Van Ginkel 2013

OECD 301D

Closed Bottle test

Inoculum: activated sludge OR river water

 

Dispersants or solvent:

Synperonic, Genapol, Tween 80/85, DCM

 

28d: 15 – 36% of ThOD

56d: 21 – 45% of ThOD

Van Ginkel & Geerts 2014

OECD 301B

CO2-evolution test

Inoculum: activated sludge

28d: 13-16% of ThOD

Desmares 2010

 Repetitive Die Away test

Inoculum: activated sludge

 

Dispersants:

Genapol PF-40

Nonylphenol.10EO.5PO

3 more additions after the first dose

 

14d: 20% of ThOD

 

21d, 28 d: 14% of ThOD

42d: 19% of ThOD

Balk & Meuwsen 1988

OECD302A

Semi-Continuous Activated sludge test

Inoculum: activated sludge

 

Dispersant: Tween 80

31d: 70 – 74% carbon removal

70d: 58 – 65% carbon removal

Van Ginkel &Geerts 2014

(Remark: All ready biodegradation tests are conducted in the presence of oxygen)