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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:
Between 30 March 2010 and 28 April 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
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:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): obtained on
29 March 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture: not applicable
- Method of cultivation:not applicable

- Storage conditions: not stated in report
- Storage length: not stated in report

- Preparation of inoculum for exposure
The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.0 g/l prior to use.
Duration of test (contact time):
28 d
Initial conc.:
13.1 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l

pH = 7.4

Solution b CaCl2 27.50 g/l
Solution c MgSO4.7H2O 22.50 g/l
Solution d FeCl3.6H2O 0.25 g/l

To 1 litre (final volume) of purified water* was added the following volumes of solutions
a – d.

10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): not applicable
- Test temperature: 21°C
- pH: The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
- pH adjusted: not stated in report
- CEC (meq/100 g): not stated in report
- Aeration of dilution water: not stated in report
- Suspended solids concentration: not stated in report
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre glass culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
- Method used to create anaerobic conditions: not applicable
- Measuring equipment:
CO2 analysis: The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser.
TOC analysis: The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.
- Other:

SAMPLING
- Sampling frequency: Days 2, 6, 8, 10, 14, 21, 28 and 29
- Sampling method: not stated in report
- Sterility check if applicable: not applicable
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Toxicity control: For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (39.3 mg) was adhered onto a glass microscope slide* prior to addition to inoculated culture medium. An aliquot (51.4 ml) of the 1000 mg/l sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 13.1 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

STATISTICAL METHODS: No statistics reported
Reference substance:
other: Sodium benzoate
Preliminary study:
No preliminary study described in report
Test performance:
The total CO2 evolution in the control vessels on Day 28 was 31.33 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
Parameter:
% degradation (CO2 evolution)
Value:
71
Sampling time:
28 d
Details on results:
The test item attained 71% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item has exhibited the potential for rapid degradation.
Results with reference substance:
Sodium benzoate attained 94% degradation after 14 days and 112% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Following the recommendations of the International Standards Organisation (ISO 1995) the test item was adheared onto a glass microscope slide prior to addition to the test medium in order to aid dispersion of the test item in the test medium.

The IC content of the test item suspension in the mineraldium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to thethods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2present in the test vessels. Therefore any additional CO2detected in the Day 29 samples originated from dissolved CO2that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of control replicates R1and R2. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2into the second absorber vessels occurred.

The toxicity control attained 45% degradation after 14 days and thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. 

Analysis of the testdia taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), gave percentage degradation values of 100% and 98% respectively for Replicates R1and R2The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2evolution occurring.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test item attained 71% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item is a UVCB and has exhibited the potential for rapid degradation.
Executive summary:

Introduction. A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

Methods. The test item, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) the test item was adheared onto a glass microscope slide prior to addition to the test medium in order to aid dispersion of the test item in the test medium.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results. The test item attained 71% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item is a UVCB and has exhibited the potential for rapid degradation.


Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 September 2017 to February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Cambridge Sewage Treatment Works, Cowley Road
- Storage: no data (collected on 26 September 2017)
- Preparation of inoculum for exposure: Sieved to 850µm to remove coarse particulates, settled and centrifuged at approximately 4000rpm for approximately 5-10 minutes. The supernatant discarded, sludge re-suspended in mineral media and centrifuged at approximately 4000rpm for approximately 5-10 minutes. This stage repeated once more then the supernatant discarded again and homogenised thoroughly by mechanical stirring (spoon). Dry sludge solids determined on the pellet produced.
- Concentration of sludge: 0.0301 g/l
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium according to the guidance
- Test temperature: 22 ± 2°C
- pH: not measured
- Aeration of dilution water: yes by carbon dioxide free air
- Suspended solids concentration: 0.0301 g/L
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Conical flasks of nominal volume 2000ml
- Number of culture flasks: 2 blanks, 2 positive control, 2 substance and 1 toxicity control
- Measuring equipment: sodium hydroxide traps measured using a Tekmar-Dohrmann Phoenix 8000 (the UV-Persulfate Analyser).
- Details of trap for CO2: 2 sodium hydroxide traps (200ml of 0.05M sodium hydroxide solution)

SAMPLING
- Sampling frequency: on day 2, 5, 8,12, 14, 19, 23, 28 and 29 (after extraxtion with 50% HCl)
- Sampling method: no data

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance:
acetic acid, sodium salt
Preliminary study:
Carbon content analysis was performed by oxidising a concentrated stock of the substance in DCM by heating it to approximately 900°C under a stream of oxygen. The CO2 produced by the resulting oxidation of the sample was detected by NDIR. The carbon content of the substance was measured as 70%
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
29 d
Details on results:
see table
Results with reference substance:
degradation > 60% within 14 days

Cumulative inorganic carbon (mg) from test

Time (days)

Reference

Test chemical

Toxicity control

1

2

1

2

0

0.00

0.00

0.00

0.00

0.00

2

9.20

4.79

-3.43

3.83

9.27

5

10.52

14.84

-1.78

8.27

10.11

8

15.50

17.99

-0.54

11.56

11.17

12

17.28

20.02

2.48

14.62

11.93

14

17.59

20.53

3.71

16.27

11.84

19

16.79

20.44

8.41

15.61

13.14

23

17.53

21.62

11.40

18.32

13.56

28

17.69

21.95

12.76

19.33

14.31

29

17.69

24.28

14.32

20.93

15.33

29

18.17

23.81

13.58

22.74

16.95

 

Percentage degradation

Time (days)

Reference

Test chemical

Toxicity control

1

2

Mean

1

2

Mean

0

0

0

0

0

0

0

0

2

31

16

23

-11

13

1

31

5

35

49

42

-6

28

11

34

8

52

60

56

-2

38

18

37

12

58

67

62

8

49

28

40

14

59

68

63

12

54

33

39

19

56

68

62

28

52

40

44

23

58

72

65

38

61

49

45

28

59

73

66

42

64

53

48

29

59

81

70

48

70

59

51

29

61

79

70

45

76

60

56

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The substance degraded by 60% over the 29 day contact period and is considered readily biodegradable
Executive summary:

In a test according OECD 301B the substance degraded by 60% over the 29 day contact period. The substance is a UVCB and although not meeting the 10-day window is considered readily biodegradable (see OECD and ECHA guidance)

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
24th - 25th March 1997
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, 0-31137 Hildesheim
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.

- Pretreatment:
The activated sludge is maintained in an aerobic condition by aeration for four hours and then homogenized with a mixer. The sludge is filtered and the filtrate (30 mL) is subsequently used to initiate inoculation.

- Concentration of sludge: 15 mg/L
- Colony forming units of the inoculum: 10E7 - 10E8 CFU/L
- Colony forming units in the test vessels: 10E5 - 10E6 CFU/L

- Water filtered: yes
- Type and size of filter used, if any: not stated
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:

TEST SYSTEM
The following test solutions were prepared in 5 L brown glass bottles as incubation vessels:
• two incubation vessels for the test item concentration (P1, P2 )
• one incubation vessel for the reference item (R1)
• two incubation vessels for the inoculum control (C1, C2 )
• one incubation vessel for the toxicity control (T1)

The necessary amounts of aqua bidest., nutrient media and inoculum were placed in each of the incubation vessel. The vessels were then connected to the system for the production of CO2 free air and aerated for 24 h.

After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles. The test and reference item concentrations were placed into the incubation vessels, the vessels made up to 3 L with CO2 free aqua bidest. and connected to the system for the production of CO2 free air.

Incubation took place in a temperature range between 22 ± 2 °C in a water bath. All vessels were stirred continuously throughout the test. On the 28th day 1 mL concentrated HCI was added to each of the vessels.

Aeration was continued for a further 24 h and on the 29th day the quantity of CO2 released in the last two gas wash bottles was determined.

- Measuring equipment:
pH-Meter, CORNING pH 240
Thermohygrograph, LAMBRECHT Tvp 3.015/3 K
Flow meter, KROHNE DUISBURG Tvp DK 800 PV

- CO2 analysis:
The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements. Back titration of the residual Ba(OHh with 0.05 N HCI was carried out three times a week during the first ten days and thereafter twice weekly. On the 28th day the pH of all solutions were measured prior to acidification.

EVALUATION
The theoretical production of carbon dioxide (ThC02 ) of the test item and functional control is calculated by the carbon content (2) and the sum formula (1), respectively.

ThCO2 [mgCO2/mg] = (C-atoms x molecular weight of CO2)/molecular weight of test or reference item (1)

ThC02 [mgC02/mg] = 3.67 x TOC [mgC/mg] (2)

0.05 N HCI titrated had been converted into mg of CO2 produced:

1 mL HCI =1.1 mg CO2 (3)

The amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the control groups.

The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production in the following equation (4):

Degradation [%] = [Net CO 2 - (production x 100)]/ ThCO 2 [mgCO 2 /3L] (4)

Validity Criteria
The study was performed according to OECD 301 BI C02 Evolution Test and GLP Guidelines.
The quality criteria were fulfilled according to the guideline:
• The total CO2 production in the blank at the end of the test was less than 40 mg/L.
• The percentage degradation of the functional control reached the pass level of ≥ 60 % by day 14.
Reference substance:
other: Sodium acetate
Preliminary study:
None conducted.
Test performance:
Not performed.
Parameter:
% degradation (CO2 evolution)
Value:
39
Sampling time:
28 d
Details on results:
The 10 % level (beginning of biodegradation) was reached after a adaptation period of 9 days. In the 10-day-window the mean biodegradation came to 21 %. The pass level of a biodegradation > 60 % was not reached.

The test item must be regarded to be not readily biodegradable in the 10-day-window and after 28 days.
Results with reference substance:
In the control group a maximum of 36.3 mg CO2/L was formed after 28 days (quality criterion: < 40 mg CO2/L after 28 days).

In order to check the activity of the test system sodium acetate was used as functional control. The percentage degradation of the functional control reached the pass level of > 60 % after 8 days. After 14 days a degradation rate of 69 % was reached. The validity criterion of the guideline is fulfilled.

Table: Biodegradation of the test item WOLLWACHSALKOHOL I LANOLINALKOHOL in comparison to the functional control and toxicity control

 

Biodegradation [%]

 

Day 6

Day 14

Day 21

 Day 28

Test Item

15 mg/L

          5

16

23

39

Functional Control 35 mg/L

 

51

 

69

 

75

 

69

Toxicity Control

15 mg/L test item + 35 mg/L reference item

 

 

26

 

 

 

49

 

 

46

 

 

50

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test material attained 39 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Executive summary:

The ready biodegradability was determined with a non-adapted activated sludge for the test item WOLLWACHSALKOHOL/LANOLINALKOHOL batch no. Eucerit 6480 over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2001-06-07 to 2001-07-06 according to OECD 301 B/C02 evolution test in the DR.U.NOACK-LABORATORIUM FÜR ANGEWANDTE BIOLOGIE.

The test item was tested in a concentration of 15 mg/L in duplicates, corresponding to a carbon content (TOC) of 12.2 mgC/L in the test vessels.

The biodegradation of the test item was followed by titrimetric analyses of the quantity of CO2 , which was produced by the respiration of bacteria. The degradation was finished on day 28 by acidification, the last titration was made on day 29, after the soluble CO2 was turned out over a period of 24 h.

The CO2 production was calculated as the percentage of total CO2 that the test item could have theoretically produced based on carbon content. Biodegradation is therefore expressed as percentage ThC02 and was calculated for each titration of CO2.

In order to check the activity of the test system sodium acetate was used as functional control. The percentage degradation of the functional control reached the pass level of > 60 % after 8 days. After 14 days a degradation rate of 69 % was reached. The validity criterion of the guideline is fulfilled.

In the toxicity control containing both test and reference item a biodegradation rate of 40 % occurred within 14 days. The biodegradation of the reference item seemed not to be inhibited by the test item in the toxicity control.

The 10 % level (beginning of biodegradation) was reached after a adaptation period of 9 days. In the 10 -day-window the mean biodegradation came only to 21 %. The pass level of a biodegradation > 60 % was not reached neither in the 10 -day-window nor after 28 days.

The test item must be regarded to be not readily biodegradable in the 10-day-window and after 28 days. The validity criteria according to the guideline are fulfilled.

Table: Biodegradation of the test item WOLLWACHSALKOHOL I LANOLINALKOHOL in comparison to the functional control and toxicity control

 

Biodegradation [%]

 

Day 6

Day 14

Day 21

 Day 28

Test Item

15 mg/L

          5

16

23

39

Functional Control 35 mg/L

 

51

 

69

 

75

 

69

Toxicity Control

15 mg/L test item + 35 mg/L reference item

 

 

26

 

 

 

49

 

 

46

 

 

50

Conclusion:

The test material attained 39 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

In a test according OECD 301B the ester degraded by 60% over the 28 day contact period and is considered readily biodegradable.

Lanolin alcohol attained 39 % degradation after 28 days and Lanolin fatty acids was readily biodegradable (71% degradation over 28 days).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information