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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:
supporting study
Study period:
04 Jul - 03 Aug 2012
Reliability:
2 (reliable with restrictions)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge from the sewage plant at Taunusstein-Bleidenstadt
- Pretreatment: The sludge was washed twice with mineral nutrient solution and aerated for four hours. Sludge was homogenized in a “Warning Blender”, filtered and rinsed with deionized water. Filtrate was used as inoculum.
- Concentration of sludge: 35 mL
- Type and size of filter used, if any: cotton filter
Duration of test (contact time):
28 d
Initial conc.:
66.2 mg/L
Based on:
test mat.
Remarks:
11 mg C/L
Initial conc.:
62.9 mg/L
Based on:
test mat.
Remarks:
10 mg C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: 20.6 to 26.3 °C (mean 22.1 °C)
- Aeration of dilution water: The aeration rate of the lest system was controlled at 4 L/h being assured by precise flowmeters

TEST SYSTEM
- Culturing apparatus: 5 litre amber carboys, closed with stoppers with tubing for gas inlet and outlet
- Method used to create aerobic conditions: mineral nutrient solution was aerated for 24 h with CO2 free compressed air
- Measuring equipment: Resting Ba(OH)2 (0.025 N) in the CO2 trap removed from the trap system was titrated with HCI (0.05 N) using phenolphthalein as an indicator. Each millilitre HCI of difference between the blank control and the test series with the test item titrated corresponds to 1.1 mg of CO2 produced (as indicated in the OECD Test Guideline 301 B). For this purpose values of titration for the 'Blank' (mean values of two parallel Blank solutions) were subtracted (CO2 production by the inoculum) from that of the test unit with the test item.
- Details of trap for CO2 and volatile organics if used:CO2 released from the test item was trapped as BaCO3 using a trap system according to the guideline. Gas outlet was connected to three CO2 absorber bottles filled with 100 mL 0.025 N Ba(OH)2 and bubbling of CO2 free compressed air through the solution was continued.

SAMPLING
- Sampling frequency: On the 4th, 7th, 12th, 18th, 25st, 28th and 29th day
- Sampling method: Periodically the CO2 absorber nearest the test vessel was removed for analysis and a new CO2 absorber was connected farest carboy. Precipitation of BaCO3 in the second CO2 trap indicated that the absorber bottle nearest the test vessel had be changed any analyzed for CO2.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (2 replicates)
- Toxicity control: yes (1 replicate)
- Reference control: yes (1 replicate)

STATISTICAL METHODS: no
Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg C/L
Parameter:
% degradation (CO2 evolution)
Value:
16
Sampling time:
28 d
Remarks on result:
other: 1st Test solution (11 mg TOC/L)
Parameter:
% degradation (CO2 evolution)
Value:
16
Sampling time:
28 d
Remarks on result:
other: 2nd Test solution (10 mg TOC/L)
Parameter:
% degradation (CO2 evolution)
Value:
83
Sampling time:
28 d
Remarks on result:
other: Functional control (sodium benzoate)
Parameter:
% degradation (CO2 evolution)
Value:
56
Sampling time:
28 d
Remarks on result:
other: Toxicity control (test item + sodium benzoate)
Results with reference substance:
From the results of the toxicity control it is assumed that there is no toxicity towards the microorganisms.

Results:

The calculated mean degradation value of the test item was 16 %. Thus, the test item may be regarded as "not readily biodegradable". The control item sodium benzoate was degraded 83 % and the "10-days-window" was met. The total CO2-evolution of the blank was 82.5 mg C0 2. Thus the test is considered to be valid. The results obtained with the toxicity control indicate that there was no toxicity of the lest item towards microorganisms at the concentration used within the test.

Table 1: Results of Test item 1

Date

Time [d]

1st Test Solution with 11 mg C/L; mg CO2 generated in the test solution, cumulative

% TOC2 (= % Biodegradation) 3)

09-Jul-2012

4

1.22

1

12-Jul-2012

7

2.76

2

17-Jul-2012

12

5.09

4

23-Jul-2012

18

8.82

6

30-Jul-2012

25

13.80

10

02-Aug-2012

28

18.13

13 4)

03-Aug-2012

29

22.15

16 5)

3) % Percentage of total CO2, the test item has generated in relation to the ThCO2

4) The test solution was stopped by the addition of 1 ml conc. HCl

5) The difference between this titration and that before is due to the liberation of CO2, after acidification of the test solution

Table 2: Results of Test item 2

Date

Time [d]

2st Test Solution with 10 mg C/L; mg CO2 generated in the test solution, cumulative

% TOC2 (= % Biodegradation) 3)

09-Jul-2012

4

0.68

1

12-Jul-2012

7

1.92

1

17-Jul-2012

12

3.53

3

23-Jul-2012

18

7.06

5

30-Jul-2012

25

12.80

10

02-Aug-2012

28

17.14

13 4)

03-Aug-2012

29

21.72

16 5)

3) % Percentage of total CO2, the test item has generated in relation to the ThCO2

4) The test solution was stopped by the addition of 1 ml conc. HCl

5) The difference between this titration and that before is due to the liberation of CO2, after acidification of the test solution

Table 3: Validity criteria

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%.

< 20%

yes

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

< 60% at day 12

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.

<40 mg/L

yes

Validity criteria fulfilled:
yes
Remarks:
see table 3 in section" Any other information on results incl tables"
Interpretation of results:
not readily biodegradable

Description of key information

The substance is not readily biodegradable (0% biodegradation after 56 d, enhanced study OECD 301 F).

Key value for chemical safety assessment

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

Additional information

Two experimental studies are available investigating ready biodegradability of the substance Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5). In both studies the pass level was not reached within 28 days therefore the OECD criteria for being readily biodegradable were not fulfilled.

The key study was conducted according the OECD 301 F and non-adapted, domestic activated sludge from a sewage treatment plant was used as inoculum. At the end of the 56-day exposure period 0% degradation based on O2 measurement was determined. Thus the substance can be considered as "not readily biodegradable". An inoculum blank, a reference control and a toxicity control were run in parallel. The control item sodium benzoate was degraded with 87%. The results obtained with the toxicity control indicate that there was no toxicity of the test item towards microorganisms at the concentration used within the test. Since the derived degradation rate was 0% after 56 d the test substance did not reach the pass level of 60% biodegradation within 28 days and it is therefore not considered to be readily biodegradable according to the OECD criteria.

The supporting study confirms the results of the key study. This GLP guideline study is available investigating the ready biodegradability of the substance according to OECD 301B (SGS Institut Fresenius GmbH, 2012). Non-adapted, domestic activated sludge from a sewage treatment plant was used as inoculum. At the end of the 29 -day exposure period 16% degradation based on CO2 measurement was determined. Thus the substance can be considered as "not readily biodegradable". The control item sodium benzoate was degraded 83 % and the "10-days­ window" was met. The results obtained with the toxicity control indicate that there was no toxicity of the test item towards microorganisms at the concentration used within the test.

In conclusion both available studies indicate that the substance is not readily biodegradable.