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Environmental fate & pathways

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:
10 May 2017 - 12 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
On day 27 the aeration of blank bottle B, the toxicity control and test item bottles A and B broke down due to a clogged air filter. Aeration was restored by replacing the filter. Breakdown was short (<1 day) and had no influence on test outcome.
Qualifier:
according to guideline
Guideline:
other: ISO International Standard 10634. "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", (1995)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Activated sludge was freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.9 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (37 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of bottles:
The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each test bottle (2 litre brown coloured glass bottles). This mixture was aerated with synthetic air overnight to purge the system of CO2.

Two test suspension bottles containing test item and inoculum were used. Two inoculum blank (containing inoculum only) bottles were used. One positive control bottle containing the reference item and inoculum was used. One toxicity control bottle containing the test item, reference item and inoculum was used.

At the start of the test (day 0), reference item and weighted amounts of test item were added to the bottles containing the microbial organisms and mineral components (test item bottle A: 44.28 mg; test item bottle B: 44.35 mg and toxicity control bottle: 44.17 mg). The volumes of suspensions were made up to 2 litres with Milli-RO water to obtain the mineral medium (1 litre mineral medium contained: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water). 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. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. The test media were excluded from light.

A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 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 approximately 1-2 bubbles per second (ca. 30-100 mL/min). The test media was excluded from light.

Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15).


Determination of CO2:
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. 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® ampoule), Merck, Darmstadt, Germany).

Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days.

Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.

On the penultimate day, the pH of all test media was measured and 1 mL of concentrated HCl (37%, Merck) was added to the test bottles. The bottles were aerated overnight to drive off CO2 present and the final titration was made. The theoretical CO2 production was calculated from the results of the TOC-analysis.


Analysis:
ThCO2, expressed as mg CO2/mg test item, was calculated from the results of carbon analysis. The amount of CO2 produced by a test item was determined
by the difference (in mL of titrant) between the experimental and blank Ba(OH)2 traps. Relative biodegradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production, based on the total carbon content of the amount of test item present in the test bottles. A figure of more than 10% biodegradation was considered significant. The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control.
Reference substance:
acetic acid, sodium salt
Preliminary study:
N/A
Test performance:
- The positive control item was biodegraded by at least 60% (84%) within 14 days.
- The difference of duplicate values for %-degradation of the test item was always less than 20 (¿ 5%).
- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (51.7 mg CO2 per 2 litres of medium, corresponding to 25.9 mg CO2/L).
- The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 15 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
29 d
Remarks on result:
other: Test item bottle A
Remarks:
Biodegradation ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
29 d
Remarks on result:
other: Test item bottle B
Remarks:
Biodegradation ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
4
Sampling time:
29 d
Remarks on result:
other: Bottle A and B mean value
Remarks:
Biodegradation ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.
Details on results:
Theoretical CO2 production:
The ThCO2 of the test item was calculated to be 2.48 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation:
Relative biodegradation values calculated from measurements performed during the test period revleaed no biologically relevant biodegradation (6% and 1% in bottle A and B, respectively) of test item.
In the toxicity control, more than 25% biodegradation occurred within 14 days (36%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.
Results with reference substance:
The reference substance sodium acetate showed 84% biodegradation, based on ThCO2 after 14 days (last measurement was on day 15, however biodegradation ended on day 14 by addition of HCl).

CO2 Production and Percentage Biodegradation of the Positive Control Item

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

Positive control

1

48.19

47.79

0.40

0.4

0.4

1

4

45.07

17.95

27.12

29.8

30.3

35

6

46.57

30.95

15.62

17.2

47.4

55

8

47.75

41.69

6.06

6.7

54.1

63

11

47.47

43.44

4.03

4.4

58.5

68

152)

46.16

34.33

11.83

13.0

71.5

84

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 mg CO2/2L.

2): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

 

 

CO2 Production and Percentage Biodegradation of the Test Item (Bottle A)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

Bottle A

1

48.19

47.16

1.03

1.1

1.1

1

4

45.07

44.62

0.45

0.5

1.6

1

6

46.57

45.86

0.70

0.8

2.4

2

8

47.75

47.26

0.49

0.5

2.9

3

11

47.47

46.91

0.56

0.6

3.5

3

15

46.16

45.82

0.34

0.4

3.9

4

18

46.33

46.03

0.30

0.3

4.2

4

22

45.51

45.36

0.15

0.2

4.4

4

25

45.99

46.55

0.00

0.0

4.4

4

292

44.75

44.13

0.62

0.7

5.1

5

292

47.66

47.03

0.63

0.7

5.8

5

292

49.85

49.29

0.56

0.6

6.4

6

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 109.8 mg CO2/2L.

2): CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

 

CO2 Production and Percentage Biodegradation of the Test Item (Bottle B)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

Bottle B

1

48.19

48.65

0.0

0.0

0.0

0

4

45.07

46.00

0.0

0.0

0.0

0

6

46.57

46.86

0.0

0.0

0.0

0

8

47.75

47.60

0.15

0.2

0.2

0

11

47.47

46.76

0.71

0.8

0.9

1

15

46.16

46.65

0.0

0.0

0.9

1

18

46.33

47.48

0.0

0.0

0.9

1

22

45.51

45.87

0.0

0.0

0.9

1

25

45.99

46.79

0.0

0.0

0.9

1

292

44.75

44.27

0.48

0.5

1.5

1

292

47.66

48.16

0.0

0.0

1.5

1

292

49.85

49.89

0.0

0.0

1.5

1

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 110.0 mg CO2/2L.

2): CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

 

 CO2 Production and Percentage Biodegradation of the Toxicity Control

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

Toxicity control

1

48.19

48.39

0.0

0.0

0.0

0

4

45.07

17.86

27.21

29.9

29.9

15

6

46.57

33.01

13.56

14.9

44.8

23

8

47.75

40.55

7.20

7.9

52.8

27

11

47.47

40.56

6.91

7.6

60.4

31

152)

46.16

37.68

8.48

9.3

69.7

36

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and positive control: 195.1 mg CO2/2L (ThCO2 test item: 109.5 mg CO2/2L + ThCO2 sodium acetate: 85.6 mg CO2/2L).

2): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

 

 

 Comparison of Biodegradation of the Test Item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

¿ A-B1)

1

1

0

1

1

4

1

0

1

1

6

2

0

1

2

8

3

0

2

3

11

3

1

2

2

15

4

1

3

3

18

4

1

3

3

22

4

1

3

3

25

4

1

3

3

292)

5

1

3

4

292)

5

1

3

4

292)

6

1

4

5

1): Absolute difference in biodegradation between bottles A and B

2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was designated as not readily biodegradable.
Executive summary:

The ready biodegradability of the test item Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was investigated in an aerobic ready biodegradability study measuring CO2 evolution according to OECD Guideline 301B.

 

Due to insufficient solubility to all preparation of an aqueous solution at a concentration of 1 g/L the test item was added by weight, in which weighed amounts of test item were added to a watch glass that was transferred to the 2-litres test bottles containing medium with microbial organisms and mineral components. Test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days (last CO2-measurement on day 29) and 14 days for the positive and toxicity control (last CO2measurement on day 15).

 

Relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation (6% and 1% in bottle A and B, respectively) of test item. The test item is therefore concluded to be not readily biodegradable.

 

The toxicity control showed that the test item did not inhibit microbial toxicity.

Description of key information

The ready biodegradability of the test item Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was investigated in an aerobic ready biodegradability study measuring CO2 evolution according to OECD Guideline 301B.The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation (6% and 1% in bottle A and B, respectively) of test item. The test item is therefore concluded to be not readily biodegradable (Timmer, 2017).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information