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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:
31 Oct - 30 Nov 2017
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)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: supernatant of activated sludge, non-adapted, domestic
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. The supernatant liquid was used as inoculum.
- Storage conditions: Mineral components, Milli- RO water and inoculum was aerated with synthetic air overnight to purge the system of CO2.
- Storage length: Over night
- Pretreatment: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was allowed to settle (41 min) and the supernatant liquid was then used as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: 4.3 g/L in the concentrated sludge.
Duration of test (contact time):
28 d
Initial conc.:
36.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline; tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon
- Test temperature: 22 - 23 °C
- pH: 7.5 - 7.9
- pH adjusted: yes
- Aeration of dilution water: aerated and stirred continuously at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Suspended solids concentration: 4.3 g/L in the concentrated sludge
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2 bottles (with test item)
- Method used to create aerobic conditions: 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 passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Measuring equipment: 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).
- Test performed in closed vessels: no
- Details of trap for CO2: emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask

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 item. Positive and toxicity control: at laest 14 days
- Sampling method: 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).

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles
- Toxicity control: 1 bottle
- Other: positive control: 1 bottle
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
28 d
Remarks on result:
other: Vessel A
Parameter:
% degradation (CO2 evolution)
Value:
20
Sampling time:
28 d
Remarks on result:
other: Vessel B
Results with reference substance:
The positive control item was biodegraded by at least 60% (73%) within 14 days.

Biological Results:

Table 1: CO2 Production and Percentage Biodegradation of the Positive Control Item

Day

HCl (0.05 N) titrated (mL)

Produced CO2

Produced CO2

Cumulative CO2

Biodegradation 1)

 

Blank (mean)

Positive Control

(mL HCl)

(mg)

(mg)

(%)

2

47.08

45.21

1.87

2.1

2.1

2

5

46.19

22.01

24.18

26.6

28.6

33

8

45.69

32.09

13.60

15.0

43.6

51

12

45.65

35.41

10.24

11.3

54.9

64

152)

46.23

39.14

7.09

7.8

62.7

73

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.8 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.

 

Table 2: CO2 Production and Percentage Biodegradation of the Toxicity Control

Day

HCl (0.05 N) titrated (mL)

 

 

Produced CO2

Produced CO2

Cumulative CO2

Biodegradation 1)

 

Blank (mean)

Toxicity control

(mL HCl)

(mg)

(mg)

(%)

2

47.08

47.10

0.00

0.0

0.0

0

5

46.19

24.46

21.73

23.9

23.9

14

8

45.69

26.14

19.55

21.5

45.4

26

12

45.65

33.67

11.98

13.2

58.6

34

152)

46.23

35.95

10.28

11.3

69.9

40

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and positive control: 173.8 mg CO2/2L (ThCO2 test item: 88.0 mg CO2/2L + ThCO2 sodium acetate: 85.8 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.

 

Table 3: Comparison of Biodegradation of the Test Item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

Δ A-B 1)

2

0

1

1

1

5

1

2

2

1

8

3

4

4

1

12

6

6

6

0

15

7

10

9

3

19

8

13

11

5

23

9

16

13

7

29 2)

11

19

15

8

29 2)

11

20

16

9

29 2)

11

20

16

9

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.

Table 3: Validity criteria for OECD 301 B.

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

9%

Yes

Percentage degradation of the reference compound has reached the pass levels by day 14.

73%

Yes

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

40%

Yes

The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.

< 5%

Yes

The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

38.6 CO2 /2 L

19.3 mg CO2/L

Yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
In conclusion, the test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Description of key information

Not readily biodegradable (11% and 20% after 28 d, OECD 301B)

Key value for chemical safety assessment

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

Additional information

One biodegradation screening study according to OECD 301 B (GLP) is available for the substance N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate (CAS 25646-71 -3)

(Timmer, 2018b). The supernatant of non-adapted, domestic activated sludge from a sewage treatment plant was used as inoculum. The test item was found to be not readily biodegradable under the test conditions at the end of the 28 -day exposure period. Two replicates were performed for the test item resulting in a degradation of 11 and 20% respectively. All validity criteria of the study were met. In conclusion, the test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.