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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:
11 October 2017 - 10 November 2017
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
Details on test material:
- Physical appearance: dark amber solid
- Storage conditions: at room temperature
Specific details on test material used for the study:
ThCO2 of the test item was calculated to be 2.53 mg CO2/mg.

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.
- Concentration of suspended solids: 3.5 g/L in the concentrated sludge.
- Preparation of inoculum for exposure: The sludge was allowed to settle (43 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- 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.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
17.5 mg/L
Based on:
test mat.
Remarks:
nominal
Initial conc.:
2.53 other: CO2/mg
Based on:
ThCO2
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONCENTRATIONS:
- Test solutions: bottle A: 17.48 mg test item per litre and bottle B 17.24 mg test item per litre

CONTROLS:
- Inoculum blank: containing only inoculum (2 bottles)
- Positive control: containing reference item and inoculum (1 bottle).
- Toxicity control: containing test item, reference item and inoculum (1 bottle).

PREPARATION OF TEST SOLUTIONS: Sine the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, small watch glasses were used to add weighed amounts to the 2-litres test bottles containing medium with microbial organisms and mineral components. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.

TEST CONDITIONS
- Composition of medium: According to OECD 301B
- Test temperature: 22-24 °C
- pH: at start: 7.6, t=14d: 7.9 (positive and toxicity control), t=28d: 7.4-7.5 (blank controls and test bottles)
- pH adjusted: no
- Test media were excluded from light and the test vessels were brown coloured glass bottles.
- Test media were aerated and stirred continuously.

TEST SYSTEM
- Number of culture flasks/concentration: 2 for test suspension, 2 for inoculum blank, 1 for positive control, 1 for toxicity control.
- Method used to create aerobic conditions: Aeration with synthetic air (CO2 < 1 ppm).
- 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).
- Details of trap for CO2 and volatile organics: 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. 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.

SAMPLING
- Sampling frequency: Titrations were made on day 2, 5, 8, 11, 15, 18, 22, 25 and 29. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Titration of Ba(OH)2 in the gas scrubbing bottles. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
40 mg/L (1.07 mg CO2/mg based on ThCO2)

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Mean of 2 bottles
Value:
5.5
Sampling time:
28 d
Details on results:
- Biodegradation: The test item biodegraded for 5% and 6% (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
- Toxicity control: More than 25% biodegradation occurred within 14 days (30%, 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.

BOD5 / COD results

Results with reference substance:
The positive control, sodium acetate, showed a normal biodegradation curve and reached 84% biodegradation after 14 days, which passes the validity criterium of at least 60% biodegradation within 14 days.

Any other information on results incl. tables

Table 1 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)

Biodegradationa) (%)

Blank (mean)

Bottle A

1

49.34

49.36

0.00

0.0

0.0

0

4

47.44

47.17

0.27

0.3

0.3

0

6

47.37

47.05

0.32

0.4

0.6

1

8

47.68

47.76

0.00

0.0

0.6

1

11

46.72

46.39

0.33

0.4

1.0

1

15

45.72

45.64

0.08

0.1

1.1

1

18

45.24

45.03

0.21

0.2

1.3

1

22

44.73

44.29

0.44

0.5

1.8

2

25

41.77

41.29

0.48

0.5

2.3

3

29b)

43.05

43.26

0.00

0.0

2.3

3

29b)

46.26

44.71

1.54

1.7

4.0

5

29b)

48.55

47.33

1.22

1.3

5.4

6

a): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.4 mg CO2/2L.

b): 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 2 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)

Biodegradationa) (%)

Blank (mean)

Bottle B

1

49.34

49.43

0.00

0.0

0.0

0

4

47.44

47.80

0.00

0.0

0.0

0

6

47.37

47.13

0.24

0.3

0.3

0

8

47.68

47.38

0.29

0.3

0.6

1

11

46.72

46.60

0.12

0.1

0.7

1

15

45.72

45.64

0.08

0.1

0.8

1

18

45.24

44.83

0.41

0.5

1.3

1

22

44.73

44.60

0.13

0.1

1.4

2

25

41.77

41.66

0.11

0.1

1.5

2

29b)

43.05

42.92

0.13

0.1

1.7

2

29b)

46.26

44.95

1.30

1.4

3.1

4

29b)

48.55

47.63

0.92

1.0

4.1

5

a): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 87.2 mg CO2/2L.

b): 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 Toxicity Control

Day

HCl(0.05 N) titrated (mL)

Produced CO2 (mL HCl)

Produced CO2 (mg)

Cumulative CO2 (mg)

Biodegradationa) (%)

Blank (mean)

Toxicity control

1

49.34

49.77

0.00

0.0

0.0

0

4

47.44

48.35

0.00

0.0

0.0

0

6

47.37

48.11

0.00

0.0

0.0

0

8

47.68

42.71

4.97

5.5

5.5

3

11

46.72

21.85

24.87

27.4

32.8

19

15b)

45.72

27.75

17.97

19.8

52.6

30

a): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and positive control: 173.7 mg CO2/2L (ThCO2test item: 88.2 mg CO2/2L + ThCO2sodium acetate: 85.4 mg CO2/2L).

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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'overall remarks'
Interpretation of results:
not readily biodegradable
Conclusions:
In a biodegradability test (Modified Sturm Test), performed according to OECD 301B and GLP principles, INCA 460: DEPDA Adduct was not readily biodegradable (5% and 6% biodegradation in duplicate bottles tested).