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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:
16 January 2018 - 15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
Appearance: Off-white powder
Test item storage: At room temperature

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: freshly obtained sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium.
- Pre-treatment: 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): 3.0 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.:
15 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 - 23°C
- pH:
at the start: 7.6
on day 14 (in the positive control and the toxicity control): 7.7
on day 28 (in the blank controls and the test solutions): 7.5-7.6
- pH adjusted: yes, at the start of the test the pH was adjusted to 7.6 using 1 M HCl
- Suspended solids concentration: the initial sludge (3.0 g/L) was used as inoculum at the amount of 5 mL/L of mineral medium, leading to a SS concentration of 15 mg/L.
- Continuous darkness: yes
- Other: The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface.

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) 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 control were made over a period of 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. 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:
Test substance (added as weighed amounts to the bottles): bottle A: 15.1 mg/L test substance; bottle B: 14.95 mg/L test substance
Reference control: 40 mg/L reference substance
Toxicity control: 14.95 mg/L test substance and 40 mg/L reference substance
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
Purity: 99.5%

Results and discussion

% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
9
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 substance was calculated to be 2.97 mg CO2/mg and that of the reference substance was calculated to be 1.07 mg CO2/mg.
- The test item degraded for 9% and 2% in duplicate bottles tested
- The criterium of at least 60% biodegradation within a 10-day window was not met. Therefore, the substance can not be considered readily biodegradable.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, 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 (90% biodegradation in 14 days)

BOD5 / COD results

Results with reference substance:
Sodium acetate showed a biodegradation of 90% after 14 days.

Any other information on results incl. tables

Table 1 CO2 Production and Percentage Biodegradation of the Reference Item

Day

HCl (0.05 N) titrated (mL)

Produced CO2 (mL HCl)

Produced CO2 (mg)

Cumulative CO2 (mg)

Biodegradation* (%)

Blank (mean)

Positive control

2

47.33

27.85

19.48

21.4

21.4

25

5

44.65

20.48

24.17

26.6

48.0

56

8

44.70

31.58

13.12

14.4

62.4

73

12

45.60

39.16

6.44

7.1

69.5

81

15#)

46.34

39.58

6.76

7.4

77.0

90

*): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.6 mg CO2/2L.
#): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

 

Table 2 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

2

47.33

46.83

0.50

0.5

0.5

1

5

44.65

43.28

1.36

1.5

2.0

2

8

44.70

43.07

1.63

1.8

3.8

4

12

45.60

44.35

1.25

1.4

5.2

6

15

46.34

45.17

1.17

1.3

6.5

7

19

46.44

45.92

0.52

0.6

7.1

8

23

46.99

46.48

0.51

0.6

7.6

8

29#

44.83

44.85

0.00

0.0

7.6

8

29#

47.42

46.77

0.65

0.7

8.3

9

29#

49.23

49.33

0.00

0.0

8.3

9

*): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 89.7 mg CO2/2L.

#): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table 3 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

2

47.33

47.30

0.03

0.0

0.0

0

5

44.65

43.49

1.15

1.3

1.3

1

8

44.70

44.53

0.17

0.2

1.5

2

12

45.60

45.88

0.00

0.0

1.5

2

15

46.34

47.16

0.00

0.0

1.5

2

19

46.44

46.77

0.00

0.0

1.5

2

23

46.99

47.36

0.00

0.0

1.5

2

29#

44.83

45.97

0.00

0.0

1.5

2

29#

47.42

48.14

0.00

0.0

1.5

2

29#

49.23

49.76

0.00

0.0

1.5

2

*): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.8 mg CO2/2L.

#): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table 4 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

2

47.33

30.06

17.27

19.0

19.0

11

5

44.65

23.59

21.06

23.2

42.2

24

8

44.70

31.58

13.12

14.4

56.6

32

12

45.60

39.05

6.55

7.2

63.8

37

15#

46.34

40.18

6.16

6.8

70.6

40

*): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and positive control: 174.4 mg CO2/2L (ThCO2test item: 88.8 mg CO2/2L + ThCO2sodium acetate: 85.6 mg CO2/2L).

#): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Table 5 Comparison of Biodegradation of the Test Item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B*

2

1

0

1

1

5

2

1

2

1

8

4

2

3

2

12

6

2

4

4

15

7

2

5

5

19

8

2

5

6

23

8

2

5

6

29#

8

2

5

6

29#

9

2

6

7

29#

9

2

6

7

*): 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:
not readily biodegradable
Conclusions:
A ready biodegradation test (Modified Sturm test), performed according to OECD 301B and GLP principles, showed that D6-Noreth was biodegraded for 9% and 2% in the duplicate bottles in 28 days. Since the pass level of 60% biodegradation within a 10-day window was not met, D6-Noreth is determined to be not readily biodegradable under the conditions of this test.