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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 25 JUL 2007 to 13 DEC 2007
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater
- Laboratory culture: no
- storage condition: aerated at room temp
- storage length: 2 days
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
- Pretreatment: Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of two days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with thest water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.
- Concentration of sludge: 30 mg/L (based on dry weight)
- Test water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Test water containing 30 mg/L activated sludge (based on dry weight)
- Preparation of test water:
The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:

a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.4.

b) MgSO4 x 7H2O 22.50 g/L

c) CaCl2 x 2H2O 36.40 g/L

d) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter

To obtain the final test water, 10 mL of stock solution a) and 1 mL each of stock solutions b and d were added to about 800 ml purified water and made up to 1000 mL with purified water. The pH of the final test water was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.

- Additional substrate: no
- Solubilising agent : On the following day (day 0), defined amounts of the test item were directly added to the test flasks. No emulsifiers or solvents were used.
- Test temperature: 21-23 °C, the inoculated flasks were incubated in a temperature-controlled room. The temperature was checked on each sampling date in a separate flask with purified water. Additionally, the room temperature was continuously recorded.
- pH: prior to the start (day 0), the pH was measured in each test flask after the addition of test and/or reference item. At the end of incubation (exposure day 28), the pH was measured again in each test flask.
- pH adjusted: no adjustment during the test
- CEC (meq/100 g): no data
- Aeration of dilution water: The test media were aerated overnight with CO2-free air to purge the system of carbon dioxide.
- Suspended solids concentration: 30 mg of dry material per liter.
- Continuous darkness: yes, the test vessels were incubated in a dark room.

TEST SYSTEM
- Culturing apparatus: test flasks: 5-L flasks (amber glass) filled with 2400-3000 ml of untreated test medium. To each flask 90 ml activated inoculum was added.
The test flasks were made up to a volume of 3 L with test water (purged with CO2-free air). 2 absorber flasks, the first one containing 300 ml 0.05 M NaOH and the second one containing 200 ml 0.05 M NaOH , were connected in series to the exit air line of each test flask.
- Number of culture flasks/concentration: 2 for test substance (30 mg/L), 2 for inoculum control (0 mg/L of TS), 2 for reference (25.7 mg/L of reference), and 1 toxicity control (30.2 mg/L of TS and 25.7 mg/L of reference substance)
- Method used to create aerobic conditions: during holding, the sludge was aerated at room temperature until use.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: CO2-free air: Air was led through a bottle containing about 750 ml of a 2 M NaOH solution to trap CO2. The CO2-free air was passed through the test solutions at a rate corresponding to about 30-100 ml/min.

SAMPLING
- Sampling frequency:
measurements have been made, at:
Exposure day 2, 5, 7, 9, 12, 14, 19, 23, 27, 28 and 29 for Test item and inoculum control
Exposure day 2, 7, 14, 28 and 29 for Procedure control
Exposure day 7, 14, 28 and 29 for Toxicity control
- Sampling method:
On each sampling day, an aliquot of 5.0 ml was withdrawn from the absorber flask nearest to the test flask for analysis of inorganic carbon (IC).
Additional samples for analysis of IC were withdrawn from the second absorber flask on exposure day 14 (around the attainment of the 10-day window) for the procedure and inoculum controls, and at the end of the exposure period on exposure day 28 for all test vessels in order to correct for any carry over of CO2. Next, 1 ml of concentrated HCl was added to each test flask and the flasks were aerated overnight with CO2-free air to drive off any residual CO2 present. On day 29, a sample from each absorber flask was withdrawn and analyzed for IC to determine residual CO2 that was present in the test suspensions on exposure day 28. In this way, any residual CO2 remaining in the test suspensions was determined as the difference between the amounts of IC found before and after acidification. At the start of the test (day 0), inorganic and total carbon (IC and TC respectively) were measured in the test flasks containing the test item (without the toxicity control) and in the inoculum controls. The IC in the test flasks containing the test item was less than 5% of the TC.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2, Containing no test and no reference substance but inoculum (see test scheme in Table 1 below)
- Abiotic sterile control: 0
- Adsorption control: 0
- Toxicity control: 1, Containing test and reference substance and inoculum (see test scheme in Table 1 below)
- other: procedure control: 2, Containing reference substance and inoculum (see test scheme in Table 1 below)

CALCULATIONS
The absolute amount of IC produced was calculated for each sampling date from the actual IC content in the absorber flasks plus the sum of the amount of IC removed in the analytical samples up to the respective sampling date. The IC content in the absorber flasks and in the analytical samples, removed from the absorber flasks, was calculated from the product of the actual IC concentration in the absorber flasks and the actual volume of absorbent or the sample volume.
IC content in absorber flask: mg IC = ICabs x VNaOH
IC removed in analytical samples: mg ICsample = IC abs x Vsample
IC produced per test flask: mg ICprod = mg IC + sum mg ICsample

Where
mg IC = IC content in absorber flask at sampling date (mg C)
IC abs = IC measured in absorber flask on sampling date (mg C/L)
VNaOH = Volume of NaOH in absorber flask on sampling date (L)
mg ICprod = Absolute amount of IC produced per test flask (mg C)
sum mg ICsample = Sum of IC removed in the analytical samples up to sampling date (mg C)

The percent degradation was calculated from:
% degradation = mg ICprod in test flask - mg ICprod in blank / mg TOC x 100%

Where
test flasks = flasks containing test item and/or reference item
blank = flasks containing neither test item nor reference item
TOC = mg TOC added as test and/or reference item

The amount of IC found in the second absorber flasks on exposure day 14 (procedure and inoculum controls) and 28 (all test flasks) was extrapolated assuming a linear increase between each sampling. The IC formation in mg C and the percentage degradation within these time periods were calculated by the sum of the amount of IC found in the first absorber flasks and the extrapolated amount in the second absorber flasks.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
no
Test performance:
The pH measured in all flasks at the end of exposure (Day 28) was between 7.3 and 7.5.
The temperature during the test period was in the range 21-23°C.
The residual amounts of CO2 found in the absorber flasks after driving off were in the range 1.0-2.6 mg C/L.
Parameter:
% degradation (inorg. C analysis)
Value:
74
Sampling time:
28 d
Remarks on result:
other: 10-d window criteria fulfilled
Details on results:
- Degradation of the test substance:
The percent biodegradationof the test item was calculated based on a total carbon content (TOC) of 0.54 mg C/mg test item.
The CO2 formation of Dimethyl 2-methyl glutarate in the test media significantly increased from exposure day 5 until test termination after 28 days. At the end of the 28-day exposure period, average biodegradation was 74%. Moreover, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was reached.

- Degradation of the toxicity control:
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total carbon content (TOC) of the test item and the reference item.
Over the 28-day exposure period, CO2 formation in the toxicity was similar but consistently higher than in the 2 procedure control, containing only the reference item. Within 14 days of exposure, biodegradation reached 57%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 30 mg/L because biodegradation in the toxicity control was > 25% within 14 days of incubation.
Results with reference substance:
The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure controls, average biodegradation of the reference item was 74% by exposure day 14, thus confirming suitability of the activated sludge (> 60% degradation by exposure day 14). By the end of the test (exposure day 28), average biodegradation was 77%.

Table 2 : Inorganic carbon (IC concentrations) measured in the absorber flasks

 

Time (days)*

IC found in absorber flasks (ICabs, mg C/L)

Test item

Na-benzoate

Toxicity control

Inoculum control

Replicate N°.

1

2

1

2

1

1

2

2/1

5/1

7/1

9/1

12/1

14/1

14/2

19/1

23/1

27/1

28/1

28/2

13.1

31.4

57.4

88.9

112.7

119.3

-

139.1

143.2

147.1

149.7

61.8

14.0

32.3

61.5

96.1

115.5

122.5

-

137.3

141.7

145.9

146.2

58.4

66.6

-

101.9

-

-

118.7

53.9

-

-

-

135.5

60.8

72.3

-

115.2

-

-

130.1

36.6

-

-

-

149.5

41.7

-

-

144.0

-

-

183.0

-

-

-

-

202.2

119.7

12.2

19.3

22.7

26.9

32.7

37.0

11.7

45.1

50.1

58.4

59.8

16.0

12.8

20.5

24.8

28.9

35.1

38.7

6.5

45.6

48.5

54.2

56.9

9.0

29/1

29/2

157.3

72.6

153.1

65.4

145.8

71.1

161.7

53.1

204.9

131.0

77.1

26.7

71.9

17.0

 

*: Absorber flask 1 or 2

-: No samples taken

Table 3 : Absolute amount of inorganic carbon (IC) produced

 

Time (days)

IC formation per test flask (ICprod, mg C/3 L)

Test item

Na-benzoate

Toxicity control

Inoculum control

Replicate N°.

1

2

1

2

1

1

2

2

5

7

9

12

14

19

23

27

28

4.8

11.5

20.0

29.8

37.8

40.5

48.1

50.9

53.7

54.8

5.0

11.7

21.0

31.7

38.4

41.1

47.2

50.0

52.8

53.3

20.0

-

35.8

-

-

46.0

-

-

-

52.2

21.7

-

38.0

-

-

46.0

-

-

-

52.5

-

-

49.2

-

-

66.7

-

-

-

84.2

4.0

6.4

7.5

8.7

10.6

13.1

14.0

15.6

18.0

18.4

4.0

6.6

8.0

9.4

11.4

12.6

13.7

14.7

16.3

17.0

29

58.8

56.4

57.0

58.1

87.2

26.3

23.1

 

-: No samples taken

Table 4 : Biodegradation of the test item and the reference item

 

Time (days)

% Degradation

Test item1

Na-benzoate1

Toxicity control1

Replicate N°.

Replicate N°.

Replicate N°.

1

2

Mean

1

2

Mean

1

2

5

7

9

12

14

19

23

27

28

1.6

10.4

25.0

42.5

54.9

56.6

69.9

73.2

74.8

75.9

2.1

10.7

27.2

46.4

56.1

58.0

68.3

71.5

73.1

73.0

1.9

10.5

26.1

44.4

55.5

57.3

69.1

72.4

73.9

74.4

35.6

-

62.5

-

-

74.0

-

-

-

76.8

39.4

-

67.4

-

-

73.8

-

-

-

77.5

37.5

-

64.9

-

-

73.9

-

-

-

77.1

-

-

44.2

-

-

57.4

-

-

-

70.9

 

 

1: Based on values presented in table 3, corrected for the inoculum controls

-: No samples taken

Validity criteria fulfilled:
yes
Remarks:
(Difference of extremes of replicate values at the end of the test: < 20%; Percentage biodegradation of the reference substance reached the level of biodegradation by 14 days; Biodegradation in the toxicity control: > 25% within 14 days)
Interpretation of results:
readily biodegradable
Conclusions:
Dimethyl 2-methyl glutarate was found to be biodegradable by 74% under the test conditions within the 28-day exposure period. Moreover, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was reached. In conclusion, dimethyl 2-methyl glutarate was found to be readily biodegradable under the test conditions within 28 days.
Executive summary:

Dimethyl 2 -methyl glutarate was investigated for its ready biodegradability in a 28 -day CO2 evolution (modified sturm) test according to EU Commision Directive 92/69/EEC C.4 C (1992) and OECD Guideline for Testing of Chemicals, N° 301 B (1992).

Dimethyl 2-methyl glutarate was found to be biodegradable by 74% under the test conditions within the 28-day exposure period. Moreover, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was reached. In conclusion, dimethyl 2-methyl glutarate was found to be readily biodegradable under the test conditions within 28 days.

In the toxicity control, containing both dimethyl 2 -methyl glutarate and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, dimethyl 2 -methyl glutarate had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 30 mg/L.

In the procedure controls, average biodegradation of the reference item was 74% by exposure day 14, thus confirming suitability of the activated sludge (> 60% degradation by exposure day 14). By the end of the test (exposure day 28), average biodegradation was 77%.

Description of key information

 Dimethyl 2-methyl glutarate was found to be biodegradable by 74% under the test conditions within the 28-day exposure period. Moreover, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was reached. In conclusion, dimethyl 2-methyl glutarate was found to be readily biodegradable under the test conditions within 28 days. 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One experimental study, scored as Klimisch 1, is available (Seyfried B., 2007) and selected as a key study.