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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Jan 2012 - 16 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
equivalent or similar to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Qualifier:
equivalent or similar to guideline
Guideline:
other: ISO 10634:1995 Water quality -- Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
C4 Acrylate
IUPAC Name:
C4 Acrylate
Details on test material:
- Name of test material (as cited in study report): MTDID 7831
- Physical state: Waxy solid
- Analytical purity: 93.96 w%
- Storage condition of test material: At room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from "Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, municipal sewage treatment plant.
- Storage conditions: Continuous aeration until use.
- Storage length: Not reported ("freshly obtained")
- Preparation of inoculum for exposure: Sludge allowed to settle 58 minutes and the supernatant liquid was used as inoculum.
- Pretreatment: None
- Concentration of sludge: 4.7 g suspended solids/L in concentrated sludge (before settling). The supernatant liquid was added at a concentration of 10 mL per L mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
35 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard mineral media as per OECD 301B. Aerated with synthetic air (ca. 20% oxygen, ca. 80% nitrogen, <1 ppm CO2) overnight to purge the media of CO2.
- Test temperature: 21.9 - 22.6 deg C
- pH: 7.6 - 7.9
- pH adjusted: No
- Aeration of dilution water: Dilution water not aerated separately
- Suspended solids concentration: 10 mL/L concentrated sludge diluted in mineral medium.
- Continuous darkness: Not reported. Test vessels were brown-colored bottles
- Other: Test media aerated and stirred continuously during the test period.

TEST SYSTEM
- Culturing apparatus: 2-liter all-glass brown colored bottles
- Number of culture flasks/concentration: 2 for test suspension; 2 for inoculum blank, 1 for reference substance, 1 for toxicity control.
- Method used to create aerobic conditions: Continuous aeration and stirring. Synthetic air was passed through 0.0125M barium hydroxide to remove any CO2 present prior to use to aerate the test system. Synthetic air was sparged through scrubbing solution at a rate of ca. 30-100 mL/min.
- Measuring equipment: CO2 production determined by titrating remaining barium hydroxide with 0.05 M standardized HCl
- Test performed in open system: yes (CO2-free synthetic air blown continuously through medium)
- Details of trap for CO2 and volatile organics if used: 100 mL of 0.0125 M Ba(OH)2) in each of three bottles connected in series to the exit air line of each exposure flask.
- Other: Test substance was not sufficiently water soluble to allow preparation of an aqueous stock solution. Weighed amounts of test substance and then 10 mL of Milli-RO water were added to weighing bottles. The weighing bottles were vigorously mixed (by vortex) and the resulting suspension was added quantitatively to the test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test substance and the microorganisms.

SAMPLING
- Sampling frequency: 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.
- Sampling method: At each sample time, the CO2 absorber nearest to the test bottle was removed for analysis. Each of the remaining two absorbers was moved one position in the direction of the test bottle and a new absorber was placed at the far end of the series. Phenolphthalein was used as a pH indicator. On the 28th day, the pH of all test solutions was measured and 1 mL of 37% HCl was added to each bottle. The bottles were aerated overnight to drive off any CO2 present. The final titrations were made on day 29.
- Sample storage before analysis: None.
- Other: The theoretical CO2 production was calculated from the molecular formula.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Contained only mineral media and inoculum
- Abiotic sterile control: None
- Toxicity control: Contained test substance at 35 mg/L and sodium acetate (reference substance) at 40 mg/L plus mineral medium and inoculum
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
> 0 - < 3
Sampling time:
28 d
Remarks on result:
other: mean biodegradation, 2%
Details on results:
-The ThCO2 of C4 acrylate was calculated to be 1.07 mg CO2/mg based on structure.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg based on structure.

The molecular formula of the test substance does not contain inorganic carbon. Since the test medium was prepared in tap-water purified by reverse osmosis, the test substance in mineral medium has < 5% of total carbon as inorganic carbon.

CO2 evolution in blanks was within limits of OECD TG301B (Table 2).

CO2 evolution for the reference substance was within the 10-day window and reached 75% by Day 14 (Table 3).

CO2 evolution in the toxicity control was 38% of ThCO2 (Table 6) on day 14. No inhibition by C4 acrylate is expected.

% biodegradation vs time plot - see Figure 1

Any other information on results incl. tables

 

Table 2:  CO2production in the blank v. untreated 0.0125 M Ba(OH)2

Day

Volume of HCl titrated (mL)

CO2 Produced (mL HCl)

CO2 Produced (mg)

Cumulative CO2(mg)

Untreated Ba(OH)2

Blank test bottles

(mean of two)

2

50.92

47.66

3.27

3.6

3.6

5

50.39

47.66

2.74

3.0

6.6

7

50.72

47.91

2.81

3.1

9.7

9

51.29

47.48

3.81

4.2

13.9

14

50.96

46.37

4.59

5.1

18.9

19

51.51

45.00

6.51

7.2

26.1

23

51.05

44.17

6.88

7.6

33.7

27

50.65

44.49

6.16

6.8

40.4

29

51.00

45.05

5.95

6.5

47.0

29

50.82

48.09

2.73

3.0

50.0

29

50.65

49.46

1.19

1.3

51.3

 

Table 3:  CO2production and percentage biodegradation of the reference substance

Day

Volume of HCl titrated (mL)¹

CO2 Produced (mL HCl)

CO2 Produced (mg)

Cumulative CO2(mg)

Percentage biodegradation²

Blank (mean)

Reference substance

2

47.66

45.52

2.14

2.3

2.3

3

5

47.66

23.87

23.79

26.2

28.5

33

7

47.91

33.46

14.45

15.9

44.4

52

9

47.48

39.21

8.27

9.1

53.5

62

14

46.37

36.63

9.74

10.7

64.2

75

1, Titrated with standard 0.05N HCl

2, Calculated as the ratio between cumulative CO2produced and the ThCO2of sodium acetate: 85.6 mg CO2/ 2 L bottle

Table 4:  CO2production and percentage biodegradation of the test substance (Bottle A)

Day

Volume of HCl titrated (mL)¹

CO2 Produced (mL HCl)

CO2 Produced (mg)

Cumulative CO2(mg)

Percentage biodegradation²

Blank (mean)

Bottle A

2

47.66

49.56

0.00

0.0

0.0

0

5

47.66

48.35

0.00

0.0

0.0

0

7

47.91

48.77

0.00

0.0

0.0

0

9

47.48

48.61

0.00

0.0

0.0

0

14

46.37

47.71

0.00

0.0

0.0

0

19

45.00

46.94

0.00

0.0

0.0

0

23

44.17

46.60

0.00

0.0

0.0

0

27

44.49

46.03

0.00

0.0

0.0

0

29

45.05

46.62

0.00

0.0

0.0

0

29

48.09

48.76

0.00

0.0

0.0

0

29

49.46

50.00

0.00

0.0

0.0

0

1, Titrated with standard 0.05N HCl

2, Calculated as the ratio between cumulative CO2produced and the ThCO2of the test substance: 73.9 mg CO2/ 2 L test bottle

 

Table 5:  CO2production and percentage biodegradation of the test substance (Bottle B)

Day

Volume of HCl titrated (mL)¹

CO2 Produced (mL HCl)

CO2 Produced (mg)

Cumulative CO2(mg)

Percentage biodegradation²

Blank (mean)

Bottle B

2

47.66

49.16

0.00

0.0

0.0

0

5

47.66

47.01

0.65

0.7

0.7

1

7

47.91

46.41

1.50

1.6

2.4

3

9

47.48

47.96

0.00

0.0

2.4

3

14

46.37

46.53

0.00

0.0

2.4

3

19

45.00

45.90

0.00

0.0

2.4

3

23

44.17

44.62

0.00

0.0

2.4

3

27

44.49

44.72

0.00

0.0

2.4

3

29

45.05

46.12

0.00

0.0

2.4

3

29

48.09

48.50

0.00

0.0

2.4

3

29

49.46

49.98

0.00

0.0

2.4

3

1, Titrated with standard 0.05N HCl

2, Calculated as the ratio between cumulative CO2produced and the ThCO2of the test substance: 74.3 mg CO2/ 2 L test bottle

 

Table 6:  CO2production and percentage biodegradation of the toxicity control

Day

Volume of HCl titrated (mL)¹

CO2 Produced (mL HCl)

CO2 Produced (mg)

Cumulative CO2(mg)

Percentage biodegradation²

Blank (mean)

Toxicity control

2

47.66

46.78

0.88

1.0

1.0

1

5

47.66

27.15

20.51

22.6

23.5

15

7

47.91

36.58

11.33

12.5

36.0

23

9

47.48

37.78

9.70

10.7

46.6

29

14

46.37

34.13

12.24

13.5

60.1

38

1, Titrated with standard 0.05N HCl

2, Calculated as the ratio between cumulative CO2produced and the sum of the ThCO2for test substance and sodium acetate: 159.6 mg CO2/ 2L test bottle (ThCO2test substance: 73.9 mg CO2/2L + ThCO2 sodium acetate: 85.6 mg CO2/2L)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
> 60% degradation for reference substance; difference in biodegradation between duplicate flasks less than 20% ; inorganic carbon content of test substance in mineral medium < 5% of the total carbon content; total CO2 evolution in blank < 40 mg/L
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
C4 acrylate biodegradation was not observed under test conditions
Executive summary:

The ready biodegradability of C4 acrylate was studied in a 28-day CO2 evolution test according to OECD method 301B. Activated sludge from a domestic sewage treatment plant was used as the source of inoculum. Brown glass bottles were filled with oxygen-saturated, CO2-free medium and spiked with one of the following: C4 acrylate at 35 mg/L (10 mg TOC/L), sodium acetate (reference substance) at 40 mg/L (12 mg TOC/L), C4 acrylate plus sodium acetate (toxicity control), or inoculum alone (inoculum blank). Evolved CO2 was titrated every second or third day during the first 10 days and thereafter at least every fifth day until the 28th day. The 28-day biodegradation of C4 acrylate was found to be 0% or 3% (mean, 2%) in the two test bottles by CO2 evolution. In the toxicity control 38% biodegradation occurred within 14 days, therefore the test substance did not inhibit microbial activity. The reference substance sodium acetate showed 75% biodegradation at 14 days demonstrating the viability of the inoculum. Based on these data, C4 acrylate was found to not to have biodegraded under the conditions of the CO2 evolution test.

This study is classified as acceptable and satisfies the guideline requirements for test method OECD301B, ready biodegradability: CO2 evolution test. The results are considered reliable without restrictions and suitable for use in Risk Assessment, Classification & Labelling, and PBT Analysis.

Results Synopsis

Percent degradability: 2%

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