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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:
02 July 2018 - 02 August 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:
liquid: viscous
Details on test material:
- Appearance: Brown viscous liquid
- Storage condition of test material: 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: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Concentration of suspended solids: 3 g/L in the concentrated sludge, 8 mg suspended solids/L in the test vessels.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and washed with mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12 mg/L
Based on:
TOC
Initial conc.:
20.5 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: According to OECD 301B
- Test temperature: 22-23 °C
- pH: at start: 7.6; t = 14 d: 7.8 and 7.7 (positive and toxicity control, resp.); t = 28 d: 7.6 (blank controls and test solutions)
- pH adjusted: yes, the pH was adjusted in all vessels to 7.6 using 1 M HCl. Except for the blank control (B), where the pH was measured to be 7.7 and not adjusted. It was concluded that this deviation had no effect on the outcome of the study because at the end of the study the pH in this bottle was 7.6 and no abnormal results were observed.
- Continuous darkness: yes
- Continuous aeration and stirring: yes
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2 for test suspension, 2 for inoculum blank, 1 for procedure control (reference item), 1 for toxicity control.
- Method used to create aerobic conditions: aeration with synthetic air (mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), CO2 < 1 ppm) at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min)
- Details of trap for CO2: 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.

CONTROLS:
- Inoculum blank: containing only inoculum
- Procedure control: containing reference item and inoculum
- Toxicity control: containing test item, reference item and inoculum

PREPARATION OF TEST SOLUTIONS:
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. TOC content of the test item was determined by TOC analysis to be 58.33%. On the day of testing weighed amounts of the test item were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (small watch glasses were used to transfer the weighed amounts of test item to the respective test bottles). The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.

SAMPLING
- Sampling frequency: Titrations were made on day 2, 5, 8, 12, 15, 19, 23 and 29 for the inoculum blank and test item. Titrations for the procedure and toxicity control were made n day 2, 5, 8, 12 and 15.
- Sampling method: titration of Ba(OH)2 in the gas scrubbing bottles.
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
Purity: 99.7%

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
64
Sampling time:
28 d
Remarks on result:
other: mean of two test bottles
Details on results:
- The ThCO2 of the test item was calculated to be 2.14 mg CO2/mg.
- The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
- Biodegradation test item: The relative biodegradation values calculated from the measurements performed during the test period revealed 67% and 61% biodegradation of the test item, for Bottles A and B, resp. (based on ThCO2). However, average biodegradation of the test item in Bottels A and B did not reach ≥60% within a 10-day window. Thus, the criterion for ready biodegradability was not met.
- Toxicity control: More than 25% biodegradation occurred within 14 days (46%, 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.
- The tempreature and pH during the exposure period were within the prescribed range of the study plan.

BOD5 / COD results

Results with reference substance:
The reference substance (sodium acetate) showed a normal biodegradation curve and reached > 60% (i.e. 74%) 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)

Biodegradation1)

(%)

Blank

(mean)

Bottle A

2

49.23

49.31

0.00

0.0

0.0

0

5

47.04

46.55

0.49

0.5

0.5

1

8

46.19

45.69

0.50

0.6

1.1

1

12

44.96

37.08

7.88

8.7

9.8

11

15

46.10

31.72

14.38

15.8

25.6

29

19

45.97

31.10

14.87

16.4

41.9

48

23

46.09

39.40

6.69

7.4

49.3

56

292)

43.03

34.39

8.64

9.5

58.8

67

292)

46.66

47.16

0.00

0.0

58.8

67

292)

47.97

49.03

0.00

0.0

58.8

67

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.2 mg CO2/2L.

2): 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)

Biodegradation1)

(%)

Blank

(mean)

Bottle B

2

49.23

49.56

0.00

0.0

0.0

0

5

47.04

47.21

0.00

0.0

0.0

0

8

46.19

46.21

0.00

0.0

0.0

0

12

44.96

29.16

15.80

17.4

17.4

20

15

46.10

34.71

11.39

12.5

29.9

34

19

45.97

33.88

12.09

13.3

43.2

49

23

46.09

40.10

5.99

6.6

49.8

57

292)

43.03

39.56

3.47

3.8

53.6

61

292)

46.66

47.44

0.00

0.0

53.6

61

292)

47.97

48.85

0.00

0.0

53.6

61

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.0 mg CO2/2L.

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

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see 'Overall remarks'
Interpretation of results:
not readily biodegradable
Conclusions:
The substance was not readily biodegradable in the CO2 Evolution Test, performed according to OECD guideline 301 B and GLP principles. The substance degraded for 67% and 61% in duplicate test bottles but did not reach >60% biodegradation within the 10-day window.
Executive summary:

In a CO2 Evolution Test was performed according to OECD guideline 301B and GLP principles, the substance was evaluated for its ready biodegrabaility. The test was performed in an aerobic aqueous medium containing activated sludge over a period of 28 days.

Due to a purity <95%, the substance's Total Organic Carbon (TOC) content was determined by TOC analysis. The TOC content was 58.33% and this value was used to calculate the ThCO2 of the substance (2.14 mg CO2/mg).

The substance was tested in duplicate at a target concentration of 20.5 mg/L, corresponding to 12 mg TOC/L. Two inoculum blanks, one procedure control (sodium acetate) and one toxicity control (substance plus sodium acetate) were included. During the 28 days exposure period, the produced CO2 was determined every second or third day for the first 10 days and at least every fifth day from day 11 to day 28. Relative biodegradation values were calculated from the CO2 production relative to the ThCO2.

The substance biodegraded for 67% and 61% in duplicate bottles tested. However, as the substance did not reach ≥60% biodegradation within a 10 -day window, the criterion for ready biodegradability was not met. Since 46% biodegradation occurred in the toxicity control within 14 days, the substance was assumed not to inhibit microbial acitivity.

All set acceptability criteria for the test were met and the study is therefore considered to be valid.

Based on the obtained results in this test, the substance is concluded to be not readily biodegrable.