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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:
13 Jun - 06 Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD, ISO and EU test guidelines in an accredited GLP laboratory
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted 1992
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
adopted 2008
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Version / remarks:
adopted 1999
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment. - Preparation of inoculum for exposure: Before use, the sludge was allowed to settle for 36 min and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.- Concentration of suspended solids: 4.0 g/L (information from the sewage treatment plant)
Duration of test (contact time):
84 d
Initial conc.:
17 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS- Composition of medium: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) was used to prepare the mineral medium as recommended by OECD guideline 301 B.- Test temperature: 21.7 - 22.7 °C- pH: 7.5 - 7.6- Suspended solids concentration: 40 mg/LTEST SYSTEM- Culturing apparatus: 2 L all-glass brown coloured bottles- Number of culture flasks/concentration: 2 bottles- Method used to create aerobic conditions: CO2-free air was bubbled through the culture vessels at 30 - 100 mL/min- Details of trap for CO2: Three CO2-absorbers (bottles filled with 100 mL 12.5 mM barium hydroxide were connected in series to the exit air line of each test bottle.SAMPLING- Sampling frequency: Titrations were made on days: 2, 5, 7, 9, 14, 19, 23, 27, 34, 41, 48, 55, 62, 69, 83 and 85. Titrations for the positive and toxicity control were made over a period of 14 days. On the 84th day, the pH of all test suspensions was measured and 1 mL of concentrated HCl was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 85.- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration.CONTROL AND BLANK SYSTEM- Inoculum blank: yes, 2 bottles- Toxicity control: yes, 1 bottle- Reference control: yes, 1 bottle
Reference substance:
acetic acid, sodium salt
Preliminary study:
Not applicable
Test performance:
The temperature recorded in a vessel with water in the same room varied between 21.7 and 22.7°C.The pH of the different test media started at 7.6 by day 75 was 7.5 for all but the positive reference which remained at 7.6
Parameter:
% degradation (CO2 evolution)
Value:
31
Sampling time:
27 d
Remarks on result:
other: Bottle A
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
27 d
Remarks on result:
other: Bottle B
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
84 d
Remarks on result:
other: Bottle A
Parameter:
% degradation (CO2 evolution)
Value:
76
Sampling time:
84 d
Remarks on result:
other: Bottle B
Details on results:
- Biodegradation of the test substance of at least 60% was not reached within a 10-day window within 28 days. Thus, the criterion for ready biodegradability was not met.- 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.- On day 23 and 27, the difference of duplicate values for % degradation of the test substance was > 20.
Results with reference substance:
The reference substance sodium acetate attained 78% degradation within 14 days.

Table 1: Biodegradation of the test substance in bottles A and B

 

Day

Bottle A

Bottle B

Mean A/B

Difference A-B

2

0

0

0

0

5

0

0

0

0

7

0

0

0

0

9

1

0

1

1

14

7

1

4

6

19

18

1

10

17

23

25

2

14

23

27

31

7

19

24

34

37

26

32

11

55

60

53

57

7

85

72

76

74

4

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
The relative biodegradation values calculated from the measurements performed during the test revealed 72 and 76% biodegradation of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate, for A and B, respectively. However, biodegradation of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate of at least 60% was not reached within a 10-day window within 28 days. Thus, the criterion for ready biodegradability was not met.
Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (with prolongation up to 84 days, last CO2-measurement on the 85thday).

 

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and the ISO International Standard 9439, 1999 and ISO Standard 10634, 1995.

 

2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was a clear colourless liquid with a purity of >98% (based on GC-FID area-%). The test substance was tested in duplicate at 17 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was calculated to be 2.59 mg CO2/mg.

 

The study consisted of six bottles:

-2 inoculum blanks (no test substance),

-2 test bottles (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate),

-1 positive control (sodium acetate) and

-1 toxicity control (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate plus sodium acetate).

 

Since 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 84 days (last CO2-measurement on the 85thday).

 

The relative biodegradation values calculated from the measurements performed during the test revealed 72 and 76% biodegradation of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate, for A and B, respectively. However, biodegradation of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate of at least 60% was not reached within a 10-day window within 28 days. Thus, the criterion for ready biodegradability was not met.

 

In the toxicity control, 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was designated as not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17 Feb - 17 Mar 1995
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Original report not available and documentation insufficient for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
not specified
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS- Aeration of dilution water: Aerated by carbon dioxide free airTEST SYSTEM- Details of trap for CO2: The vessels were vented via sodium hydroxide traps which trapped any carbon dioxide produced. SAMPLING- Sampling frequency: on day 4, 7, 11, 17, 21 and 28
Reference substance:
other: sodium acetate
Parameter:
% degradation (CO2 evolution)
Value:
63
Sampling time:
28 d
Details on results:
The test substance is not readily biodegradable, but biodegradable under conditions tested. The biodegradation was > 60% within 28 days, but the 10 -day-window was not fulfilled.
Results with reference substance:
The reference substance was degraded to 69% by day 11 and to 92% by day 28.

Table 1: Percentage biodegradation (measured Carbon Dioxide/Theoretical Carbon Dioxide) of test and reference substance

Day

Test substance [%]

Reference substance [%]

4

0

11

7

8

56

11

25

69

17

51

91

21

46

80

28

63

92

 

Validity criteria fulfilled:
not specified
Interpretation of results:
other: biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Jul - 4 Aug 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance to OECD, ISO and EU test guidelines in an accreditied GLP laboratory
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted in 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide 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: Activated sludge was obtained from a municipal sewage treatment plant: "Watershap Aa en Maas", s-Hertogenbosch (The Netherlands), which predominantly treats domestic sewage.- Method of cultivation: The freshly obtained sludge was kept under continuous aeration until further treatment.- Preparation of inoculum for exposure: Before use the sludge was allowed to settle (52 min) and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
29 d
Initial conc.:
17 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS- Composition of medium: according to guideline- Test temperature: 21.8 - 22.5 °C (continuously measured in a vessel with Milli-RO water in the same room)- pH: 7.6 - 8.1 (measured at the start and the end of the test)- pH adjusted: no- Aeration of dilution water: Mineral components, Milli-RO water and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.- Suspended solids concentration: 4 g/L- Other: Since the test substance was not sufficiently soluble to allow preparations of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2 L test bottles containing medium with microbial organisms and mineral components (test substance: bottle 1: 34.6 mg, bottle 2: 34.1 mg, toxicity control: 34.5 mg). To this end, 20 mL of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test.TEST SYSTEM- Culturing apparatus: 2 L all-glass brown coloured bottles- Number of culture flasks/concentration: 2- Method used to create aerobic conditions: A mixture of oxygen (~ 20%) and nitrogen (~ 80%) was passed through a bottle containing 0.5 - 1 L 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of ~ 1-2 bubbles per second (ca. 30-100 mL/min).- Measuring equipment: 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). - Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.- Other: On day 28 the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%) was added to the bottles of the inoculum blank and test suspensions. The bottles were aerated overnight to drive off CO2 present in the test suspensions. The final titration was made on day 29.SAMPLING- Sampling frequency: 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 suspensions. Titrations for the positive and toxicity control were made at least 14 days.- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.CONTROL AND BLANK SYSTEM- Inoculum blank: 2- Abiotic sterile control: 2- Toxicity control: 1
Reference substance:
acetic acid, sodium salt
Remarks:
12 mg/L as TOC
Preliminary study:
Not applicable
Parameter:
% degradation (CO2 evolution)
Value:
>= 27 - <= 45
Sampling time:
29 d
Details on results:
The test substance was biodegradad significantly (27 and 45%) during the test period. However, since the pass level of 60% biodegradation within 28 days was not reached, the substance is not readily biodegradable according to OECD criteria under the conditions of the test.
Results with reference substance:
The reference substance was biodegraded by > 60% (77%) within 14 days.

The theoretical CO2 production of the test substance was calculated to be 2.59 mg CO2/mg.

In the toxicity control more than 25% biodegradation occured withion 14 days (40%, based on ThCO2). Therefore, the test substance is assumed not to inhibit microbial activity.

Table 1: CO2 production and percentage biodegradation of reference, treatment and toxicity control

Treatment

Day

HCl (0.05 N) titrated [mL]

Produced CO2 [mL HCl]

Produced CO2 [mg]

Cumulative CO2 [mg]

Biodegradation [%]

Blank (mean)

Positive control

reference

3

46.67

24.99

21.68

23.8

23.8

28

6

46.45

26.04

20.41

22.5

46.3

54

8

46.19

36.61

9.58

10.5

56.8

66

10

46.89

42.07

4.82

5.3

62.1

73

14

46.09

42.42

3.67

4.0

66.2

77

Test substance 

bottle 1/2

3

46.67

45.88/45.36

0.79/1.31

0.9/1.4

0.9/1.4

1/2

6

46.45

45.65/44.69

0.80/1.76

0.9/1.9

1.7/3.4

2/4

8

46.19

46.06/46.04

0.13/0.15

0.1/0.2

1.9/3.5

2/4

10

46.89

46.53/44.61

0.36/2.28

0.4/2.5

2.3/6.1

3/7

14

46.09

45.31/45.01

0.78/1.08

0.9/1.2

3.1/7.2

4/8

17

47.58

38.87/43.44

8.71/4.14

9.6/4.5

12.7/11.8

14/13

20

45.93

40.08/38.30

5.85/7.63

6.4/8.4

19.2/20.2

21/23

24

45.99

41.85/38.84

4.14/7.15

4.6/7.9

23.7/28.0

26/32

29

42.98

42.69/35.26

0.28/7.72

0.3/8.5

24.0/36.5

27/41

29

46.13

45.98/43.71

0.15/2.42

0.2/2.7

24.2/39.2

27/44

29

47.24

46.96/46.53

0.28/0.71

0.3/0.8

24.5/40.0

27/45

Toxicity control

3

46.67

24.70

21.97

24.2

24.2

14

6

46.45

27.42

19.03

20.9

45.1

26

8

46.19

36.65

9.54

10.5

55.6

32

10

46.89

41.17

5.72

6.3

61.9

35

14

46.09

39.01

7.08

7.8

69.7

40

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
The relative biodegradation values calculated from the measurements performed during the test period revealed 27 and 45% biodegradation of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate , for the duplicate bottles tested. Thus the criterion for ready biodegradability (at least 60% biodegradation within a 10 day window) was not met. In the toxicity control more than 25% biodegradation occurred within 14 days (40% based on the ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide evolution test (Modified Sturm test) with 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate.

The study procedures were described were based on the OECD test guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) 440/2008 of 30h May 2008, Publication No. L142, Part C.4-C and the ISO international standard 9439, 1990.

2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was a clear colourless liquid with a purity of at least 98% (based on GC-FID area-%). The test substance was tested in duplicate at 17 mg/L corresponding to 12 TOC/L. the organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was calculated to be 2.59 CO2/mg.

The study consisted of six bottles:

2 inoculum blanks (no test substance)

2 test bottles (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate.

1 positive control (sodium acetate) and

1 toxicity control (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate plus sodium acetate).

Since 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 20 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29thday).

 

The relative biodegradation values calculated from the measurements performed during the test revealed 27 and 45% biodegradation of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate, for the duplicate bottles tested. Thus the criterion for ready biodegradability (at least 60% biodegradation within a 10 day window) was not met.  In the toxicity control 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoat was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate was designated as not readily biodegradable.

Description of key information

 Inherently biodegradable, not fulfilling specific criteria: >20% CO2 evolution in 28 days (OECD 301B) and 74% CO2 evolution in 85 days (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS No. 28510-23-8) is not readily biodegradable according to OECD criteria. Based on one GLP study according to OECD 301B a degradation of ≥ 20% after 28 days can be assumed (Desmares-Koopmans, 2010). As stated in ECHA Guidance on information requirements and chemical safety assessment, Chapter 7b, p 180 “Biodegradation above 20% of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability.” Therefore, 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS No. 28510-23-8) can be regarded as inherently biodegradable, not fulfilling specific criteria.

Another GLP study according to OECD 301B was prolonged up to 85 days (Desmares-Koopmans, 2012). In this study the pass level (60% CO2 evolution) was reached between days 62 to 69. In both tests (Desmares-Koopmans, 2010; 2012), a prolonged lag phase of 14 days or longer was shown before degradation of the test substance started. The toxicity controls showed that no inhibition of aquatic microorganisms occurred. Based on these two tests, it can be assumed, that microorganisms need to adapt their enzymes in order to degrade 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS No. 28510-23-8). Nevertheless once these are adjusted, good overall degradation rates can be achieved (overall degradability after 85 days = 74%). Therefore in accordance with Regulation (EC) No. 1907/2006, the substance is regarded as not persistent (not P).

Due to the high overall degradation rates as observed in the enhanced biodegradation test (Desmares-Koopmans, 2012) ultimate and complete degradation can be assumed for this substance. Consequently, further simulation testing for water and sediment or soil biodegradation tests are not necessary.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within the CSR.