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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2009-07-30 - 2009-08-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD). For justification of read-across within the category please see chapter 1 of the Chemical Safety Report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to Chemikaliengesetz and Directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Test item: Licowax S
Active ingredient: Montanic acids / Montanic acid esters (genuin)
Appearance: Pale yellow powder, solid
Water solubility: Approx. 15 mg/L (room temperature)
pH value: Slightly acidic
Melting point: Approx. 83 - 84 °C (drop point)
TOC (as determined by test facility prior to study initiation): 74.0 %
Recommended storage Cool and dry:
Storage at test facility: Refrigerator 6 ± 2 °C, protected from moisture and light
Retention of test item: At least 1 g has been retained
Identification parameter at test facility: Name, batch number, state, consistency and colour
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Inoculum of the aqueous phase of non adapted activated sludge.
Source:
Municipal sewage treatment plant, D-31137 Hildesheim. Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly industrial chemical waste.
Pretreatment:
The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air for 5 days. 10 mL/L were used to initiate inoculation.

Colony forming units in the test vessel: 10(E)7 - 10(E)8 CFU/L




Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
15 mg/L
Based on:
test mat.
Initial conc.:
11.1 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test vessels: 5000 mL, brown glass
Volume of the test medium: 3000 mL
Test medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
Test temperature: 21.5 - 23.0 °C
Dispersion treatment: Continuous stirring
Aeration: 30 - 100 mL/min
Photoperiod: Low light conditions

The concentration of the test item and the theoretical CO2 production (ThCO2) were calculated based an the determined carbon content.
The following incubation vessels were prepared:
¿ two for the test item (P1, P2)
¿ one for the reference item (R1)
¿ two for the inoculum control (C1, C2)
¿ one for the toxicity control (T1)
The necessary amounts of bidistilled water, mineral salts medium and inoculum were placed in each of the incubation vessels. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
Test and reference item were weighed out. A defined volume of bidistilled water was added to the test item and it was treated with ultrasound. The test item dispersions and the reference item were transferred into the incubation vessels, the vessels were made up to 3 L with bidistilled water and connected to the system for the production of CO2 free air.
On day 28, 1 mL 37 % HCI was added to each of the vessels. Aeration was continued for further 24 h and an day 29 the quantity of CO2 released in the last two gas wash bottles was determined.
The room temperature was recorded continuously throughout the test by a thermohygrograph.

ThCO2 [mgCO2/mg] = 3.67 TOC [mgC/mg test item]

ThCO2 [mgCO2/mg] = [C- Atoms ¿ molecular weight of CO2]/[molecular weight of reference item]

The produced CO2 was calculated by:
1 mL HCI (c = 0.05 mol/L) = 1.1 mg CO2

The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Not applicable
Test performance:
Not applicable
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
8
Sampling time:
28 d
Details on results:
Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined at test start by standard dilution plate count:
2.1 x 109 CFU/L
Both test item replicates did not reach the 10 % level (beginning of biodegradation) until day 28. The mean biodegradation rate after 28 days was 8 % (6 and 9%).
In the toxicity control a biodegradation rate of 41 % was determined within 14 days and it came to 45 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

BOD5 / COD results

Results with reference substance:
The adaptation phase of the functional control changed after 3 days into the degradation phase (degradation >=10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % after 9 days and came to a maximum of 95 %, after 28 days. The validity criterion degradation equal or lager 60 % after 14 d is fulfilled

Any other information on results incl. tables

CO2-Production and Biodegradation after 28 Days

CO2-Production

Functional

Test Item

Toxicity Control

after 28 d

Control

15mg/L

15mg/L Test Item +

20 mg/L

No.1

No. 2

20 mg/L Reference Item

Net      [mg/3 L]

121.1

6.8

10.8

113.5

           [mg/L]

40.4

2.3

3.6

37.8

Theor.  [mg/3 L]

127.8

122.4

122.4

250.2

           [mg/L]

42.6

40.8

40.8

83.4

Degradation [%]
after 28 d

95

6

9

45

CO2-Production and Biodegradation in the Inoculum Control, Functional Control and Toxicity Control Samples

Study Day

Date

Inoculum
Control

Functional Control

20 mg/L

Toxicity Control

15mg/L Test Item +
20 mg/L Reference Item

 

[mg CO2/3L]

[mg CO2/3L]

Degr.

[mg CO2/3L]

Degr.

 

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

 

1

31.07.09

6.0

8.9

2.9

2

12.7

6.7

3

4

03.08.09

13.8

40.5

29.6

23

54.3

47.2

19

6

05.08.09

12.6

34.3

51.3

40

45.5

80.1

32

8

07.08.09

13.2

37.1

75.2

59

26.3

93.2

37

11

10.08.09

13.2

29.7

91.7

72

20.4

100.4

40

14

13.08.09

18.0

26.8

100.5

79

19.6

102.0

41

18

17.08.09

21.6

29.9

108.8

85

24.1

104.5

42

21

20.08.09

19.6

23.3

112.5

88

21.2

106.1

42

25

24.08.09

20.4

26.7

118.8

93

22.6

108.3

43

28

27.08.09

19.1

18.6

118.8

93

22.6

111.8

45

  29*

28.08.09

14.4

16.1

121.1

95

16.1

113.5

45

CO2-Production and Biodegradation in the Inoculum Control and Test Item Samples

Study Day

Date

Inoculum

Test Item15mg/L

Control

Replicate 1

Replicate 2

[mg CO2/3L]

[mg CO2/3L]

Degr.

[mg CO2/3L]

Degr.

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

1

31.07.09

6.0

5.2

0.0

0

5.0

0.0

0

4

03.08.09

13.8

18.4

4.6

4

12.1

0.0

0

6

05.08.09

12.6

10.8

4.6

4

11.2

0.0

0

8

07.08.09

13.2

9.5

4.6

4

12.0

0.0

0

11

10.08.09

13.2

11.7

4.6

4

12.2

0.0

0

14

13.08.09

18.0

15.4

4.6

4

14.7

0.0

0

18

17.08.09

21.6

19.9

4.6

4

21.3

0.0

0

21

20.08.09

19.6

18.7

4.6

4

21.7

2.1

2

25

24.08.09

20.4

21.0

5.2

4

23.3

5.0

4

28

27.08.09

19.1

17.8

5.2

4

22.6

8.5

7

  29*

28.08.09

14.4

16.0

6.8

6

16.7

10.8

9

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
In a ready biodegradability test (CO2-evolution) according to relevant guidelines and compliant to GLP (reliability category 1), the test item proved to be not readily biodegradable (8% biodegradation within 28 days). Since the toxicity control showed 41% degradation within 14 days and 45% degradation within 28 days, toxicity of the test item is not to be regarded as relevant.
Executive summary:

The ready biodegradability of the test item Licowax S was determined with a non adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2009-07-30 to 2009-08-28 according to OECD 301 B at DR.U.NOACK-LABORATORIEN. The test item was tested at a concentration of 15 mg/L in duplicates, corresponding to a carbon content (TOC) of 11.1 mg C/L in the test vessels. The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29, after residual CO2 had been purged from the test solutions over a period of 24 h. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation rate was calculated for each titration time.

To check the activity of the test system, sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % after 9 days and came to a maximum of 95 % after 28 days.

In the toxicity control containing both test and reference item a biodegradation rate of 41 % was determined within 14 days and it came to 45 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Both test item replicates did not reach the 10 % level (beginning of biodegradation) until day 28. The mean biodegradation rate after 28 days was 8 %.

The test item has to be regarded as not readily biodegradable in the 10-d-window and after 28 days.