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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 July 2017 - 04 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to
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"
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white powder with lumps
- Test item storage: at room temperature protected from light
Specific details on test material used for the study:
Solubility in water: poor

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: not applicable; freshly obtained sludge was used immediately.
- Preparation of inoculum for exposure: before use, the sludge was allowed to settle for 44 minutes.
- Pretreatment: 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 bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Initial concentration of sludge (suspended solids): 3.6 g/L
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12 mg/L
Based on:
TOC
Initial conc.:
19 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: mineral medium according to OECD 301B
- Test temperature: 22.0 - 23.1°C
- pH:
at the start: 7.6
on day 14: 7.9 and 8.0, in the positive control and the toxicity control, respectively
on day 28: 7.8, in the blank controls and the test item groups
- pH adjusted: yes, using 1 M HCl
- Suspended solids concentration: not indicated; the supernatant liquid of settled sludge was used at the amount of 10 mL/L of mineral medium.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: synthetic air (CO2 < 1 ppm) was sparged through scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Measuring method: produced CO2 reacted with barium hydroxide Ba(OH)2 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.

SAMPLING
- Sampling frequency: 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 suspension. Titrations for the positive and toxicity controls were made over a period of at least 14 days.
- Sampling method: at each sampling 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. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, containing only inoculum (2 bottles)
- Abiotic sterile control: no
- Toxicity control: yes, containing test item, reference item and inoculum (1 bottle).
- Other: positive control: containing reference item and inoculum (1 bottle).

STATISTICAL METHODS: no statistics were used

TEST CONCENTRATIONS:
- Preperation of test solutions: weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. 10 mL of Milli- RO water was added to each weighing bottle containing the test item. The test item was shortly treated with a mortar and pestle to aid weighing and to obtain a fine dispersion to increase availability. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test.
Test item (added as weighed amounts to the bottles): bottle A: 19.00 mg/L test item; bottle B: 19.10 mg/L test item
Reference control: 40 mg/L reference substance
Toxicity control: 19.04 mg/L test substance and 40 mg/L reference substance
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
Purity: 99.1%

Results and discussion

% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
15
Sampling time:
28 d
Remarks on result:
other: Bottle A
Key result
Parameter:
% degradation (CO2 evolution)
Value:
2
Sampling time:
28 d
Remarks on result:
other: Bottle B
Details on results:
- The theoretical CO2 production of the test item was calculated to be 2.29 mg CO2/mg and that of the reference substance was calculated to be 1.07 mg CO2/mg.
- The test item degraded for 15% and 2% in duplicate bottles, respectively, thus the criterium for ready biodegradability of at least 60% biodegradation was not reached.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (48%, 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 was biodegraded for 85% in 14 days and showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:
Sodium acetate showed a biodegradation of 85% after 14 days.

Any other information on results incl. tables

Table 1 CO2 production in the blank

Day

HCl (0.05 N) titrated (mL)

Produced CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Ba(OH)2#

Blank (mean)

1

50.04

47.67

2.36

2.6

2.6

4

50.43

46.46

3.98

4.4

7.0

6

50.72

47.35

3.37

3.7

10.7

8

51.17

47.04

4.13

4.5

15.2

11

51.34

47.33

4.01

4.4

19.6

15

50.56

46.48

4.09

4.5

24.1

18

50.81

46.72

4.09

4.5

28.6

22

51.10

46.45

4.65

5.1

33.7

25

50.13

45.59

4.54

5.0

38.7

29*)

50.52

44.81

5.72

6.3

45.0

29*)

49.96

46.69

3.28

3.6

48.6

29*)

49.89

48.10

1.79

2.0

50.6

#): "Strength" of untreated 0.0125 M Ba(OH)2 solution

*): 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.

Table 2 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)

Biodegradation#)

(%)

Blank

(mean)

Positive

control

1

47.67

48.11

0.00

0.0

0.0

0

4

46.46

26.70

19.76

21.7

21.7

25

6

47.35

32.93

14.42

15.9

37.6

44

8

47.04

37.32

9.72

10.7

48.3

56

11

47.33

37.57

9.76

10.7

59.0

69

15*)

46.48

33.98

12.50

13.7

72.8

85

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

*): CO2 measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

 

Table 3 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)

Biodegradation#)

(%)

Blank

(mean)

Bottle A

1

47.67

48.30

0.00

0.0

0.0

0

4

46.46

46.33

0.13

0.1

0.1

0

6

47.35

46.61

0.73

0.8

0.9

1

8

47.04

46.69

0.34

0.4

1.3

2

11

47.33

45.98

1.35

1.5

2.8

3

15

46.48

44.66

1.82

2.0

4.8

6

18

46.72

44.88

1.84

2.0

6.8

8

22

46.45

46.09

0.36

0.4

7.2

8

25

45.59

44.07

1.52

1.7

8.9

10

29*)

44.81

41.99

2.82

3.1

12.0

14

29*)

46.69

45.93

0.76

0.8

12.8

15

29*)

48.10

47.75

0.34

0.4

13.2

15

#): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 87.0 mg CO2/2L.

*): 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.

 

Table 4 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)

Biodegradation#)

(%)

Blank

(mean)

Bottle B

1

47.67

48.71

0.00

0.0

0.0

0

4

46.46

46.21

0.24

0.3

0.3

0

6

47.35

46.94

0.41

0.4

0.7

1

8

47.04

47.48

0.00

0.0

0.7

1

11

47.33

47.23

0.10

0.1

0.8

1

15

46.48

46.00

0.47

0.5

1.3

2

18

46.72

46.48

0.24

0.3

1.6

2

22

46.45

46.46

0.00

0.0

1.6

2

25

45.59

45.87

0.00

0.0

1.6

2

29*)

44.81

44.38

0.42

0.5

2.1

2

29*)

46.69

47.11

0.00

0.0

2.1

2

29*)

48.10

48.55

0.00

0.0

2.1

2

#): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 87.5 mg CO2/2L.

*): 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.

 

Table 5 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)

Biodegradation#)

(%)

Blank

(mean)

Toxicity

control

1

47.67

48.99

0.00

0.0

0.0

0

4

46.46

37.96

8.50

9.3

9.3

5

6

47.35

27.92

19.43

21.4

30.7

18

8

47.04

31.74

15.30

16.8

47.5

28

11

47.33

32.94

14.39

15.8

63.4

37

15*)

46.48

28.99

17.49

19.2

82.6

48

#): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and positive control: 172.8 mg CO2/2L (ThCO2test item: 87.2 mg CO2/2L + ThCO2sodium acetate: 85.6 mg CO2/2L).

*): 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.

Table 6 Comparison of biodegradation of the test item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B#)

1

0

0

0

0

4

0

0

0

0

6

1

1

1

0

8

2

1

2

1

11

3

1

2

2

15

6

2

4

4

18

8

2

5

6

22

8

2

5

6

25

10

2

6

8

29*)

14

2

8

12

29*)

15

2

9

13

29*)

15

2

9

13

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

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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see 'overall remarks'
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item biodegraded for 15% and 2% in duplicate bottles in 28 days. Since the pass level of 60% biodegradation was not met, the test item is considered to be not readily biodegradable under the conditions of this test.
Executive summary:

In a Modified Sturm Test, according to OECD guideline 301 B and GLP principles, ROC-118 was assessed for its ready biodegradability. The test item was tested in duplicate at a target concentration of 19 mg/L, corresponding to 12 mg TOC/L. The exposure period was 28 days for the two inoculum blanks and the test item, and 14 days for the positive control (sodium acetate) and the toxicity control. The test item was not sufficiently soluble to prepare an aqueous solution of 1 g/L. Therefore, weighed amounts were added to test bottles containing medium with microbial organisms and mineral components. To accomplish that, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. The test item was shortly treated with a mortar and pestle to aid weighing and to obtain a fine dispersion to increase availability. After vigorous mixing, the suspension was added to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms.

CO2 measurements showed that the test item biodegraded for 15% and 2%, in the duplicate bottles tested. Since the pass level for ready biodegradability of 60% was not reached, the test item was considered to be not readily biodegradable under the conditions of this test.

The toxicity control showed that the test item did not inhibit microbial activity (degradation >25%). All validity criteria were met, thus the study was considered to be valid.