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Environmental fate & pathways

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:
2016-03-31 to 2016-05-20
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Test system Non-adapted activated sludge
Receipt: 2016-03-24
Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
Municipal sewage treatment plant, 31137 Hildesheim, Germany

The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration until test start. The amount of activated sludge used to initiate inoculation was 27 mg/L dry weight (corresponding to 19.9 mL activated sludge per test vessel).
Colony forming units in the test vessel: Approx. 10exp7 - 10exp8 CFU/L

Duration of test (contact time):
49 d
Initial conc.:
10.2 mg/L
Based on:
TOC
Initial conc.:
14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test

- Solubilising agent for a better bioavailability (type and concentration if used): Silicon oil (Sigma-Aldrich), Batch No. MKBW3805V, CAS No. 63148-62-9, Received 2016-02-25, Expiry date 2018-02-25, (acc. to test facility SOP) 1 mL/L test medium were pipetted, final concentration 3 mL in the test vessel
- Test temperature: 22 ± 2 °C
- pH: Start: not mentioned; End prior to Acidification: Day 49: 7.51 - 7.62
- pH adjusted: no
- Continuous darkness: Low light conditions (brown glass bottles)
- Other: Dispersion treatment: Continuous stirring, Aeration 30 - 100 mL/min


TEST SYSTEM
- Culturing apparatus and test procedure: 5-Liter test culture vessels each containing 3 litres of solution. The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. 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. The test item and the reference item were transferred to the respective incubation vessels. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air. On day 49, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined. The room temperature was recorded continuously throughout the test. Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). For each titration the first gas wash bottle was removed and a new bottle was connected to the last one. Back titration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly.
On day 49 the pH of all solutions was measured prior to acidification.

Pretreatment: Test item weighed out into a GC-Vial (20 mL) and 1mL silicon oil/L test medium were pipetted onto the test item. Then vortexing for one minute until the test item and the silicon oil result in a homogenous solution. One GC-Vial was prepared for each replicate. This was prepared one day in advance and then stored until test start at room temperature in the closed vials.

- Number of culture flasks/concentration: 6 with test substance, 2 with silicon oil control (Silicon oil without test item, but treated as described above, Test medium without test and/or reference item), 2 with only inoculum (blank) (Test medium without test and/or reference item), 1 as toxicity control (Test item (incl. silicon oil and pre-treatment as described above) and reference item in test concentration), 1 functional control (reference control)
- Method used to create aerobic conditions: The test system was aerated with CO2-free air at a rate of 30-100 mL/min


SAMPLING
- Sampling frequency: day 1, 4, 6, 8, 11, 14, 18, 22, 25, 28, 32, 34, 39, 42, 47, 49 and 50 after acidification.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (in duplicate)
- Toxicity control: yes
- Other: reference substance used was sodium benzoate

CALCULATIONS:
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content (1) and the molecular formula (2), respectively.

ThCO2 [mgCO2/mg] = 3.67 · TOC [mgC/mg test item] (1)

ThCO2 [mgCO2/mg] = C-Atoms x molecular weight of CO2/molecular weight of reference item (2)

The produced CO2 was calculated by (3):

1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2 (3)

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 was calculated from the ratio theoretical CO2 production to net CO2 production in the following equation (4):


Degradation [%] = net CO2 x 100/ ThCO2/[mg CO2/3 l ]




Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
34 d
Remarks on result:
other: mean of replicates
Remarks:
inherently biodegradable (without pre-adaptation) and with the potential for complete mineralization
Parameter:
% degradation (CO2 evolution)
Value:
41
Sampling time:
28 d
Remarks on result:
other: mean biodegradation
Parameter:
% degradation (CO2 evolution)
Value:
88
Sampling time:
49 d
Remarks on result:
other: mean biodegradation
Parameter:
% degradation (CO2 evolution)
Value:
> 60
Sampling time:
28 d
Remarks on result:
other: 1 of 6 replicates
Results with reference substance:
The percentage degradation of sodium benzoate reached the pass level of 60% within 6 days (61 %) and a maximum biodegradation of 88% after 49 days.

In the toxicity control containing both test and reference item a biodegradation of 46 % was determined within 14 days and it came to 68% after 28 days and 80% after 49 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Validity criteria fulfilled:
yes
Remarks:
The differences between replicates rely on bioavailability, five of the six replicates are clearly within 20 % from each other.
Interpretation of results:
other: inherently biodegradable (without pre-adaptation) and with the potential for complete mineralization.
Conclusions:
The test item 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester is classified as not readily biodegradable, but inherently biodegradable (without pre-adaptation) and with the potential for complete mineralization.
Executive summary:

The ready biodegradability of the test item 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester was determined with a non-adapted activated sludge over a test period of 49 days in the Modified Sturm Test. The study was conducted from 2016-03-31 to 2016-05-20 according to OECD 301 B at the test facility. The test item was tested at a concentration of 14 mg/L with 6 replicates corresponding to a carbon content (TOC) of 10.2 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C. 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 49 by acidification of the test solutions. The last titration was made on day 50 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation 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% within 6 days and a maximum biodegradation of 88% after 49 days. In the toxicity control containing both test and reference item a biodegradation of 46 % was determined within 14 days and it came to 68% after 28 days and 80% after 49 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The mean of test item replicates reached the 10 % level (beginning of biodegradation) within 21 days. The 60 % pass level was reached by the mean of replicates on day 34. The mean biodegradation on day 28 was 41% and at test end on day 49 88%. This proves the potential for complete mineralization. Due to the difficult nature of the test item regarding bioavailability, six replicates with identical pre-treatment were used. Five replicates showed similar behaviour. These five replicates reached a mean of 47 % biodegradation after 28 days (one replicate at 60%), but exhibiting a lag phase of at least 14 days. After the lag phase, the biodegradation was rapid. One replicate was lagging behind. Although thoroughly care was taken during the pre-treatment, minimal differences seem to have great influence on the outcome. This indicates, that bioavailability is limiting for the biodegradation and not the test item’s potential for biodegradability.

Description of key information

The ready biodegradability of the test item 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester was determined with a non-adapted activated sludge over a test period of 49 days in the Modified Sturm Test. The test item 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester is classified as not readily biodegradable, but inherently biodegradable (without pre-adaptation) and with the potential for complete mineralization.

The structurally related substance, 1,2,3 -Propanetricarboxylic acid, 2 -Hydroxy-,tris(C14 -C15 -alkyl)esters, was readily biodegradable in an OECD 301B-test.

The source substance 1, Tri (hexyl, octyl, decyl)-citrate, has a mean biodegradability in the CO2 Evolution Test according to OECD 301 B of 67% after 28 days.  For UVCB substances the 10-day window condition should not be applied. Therefore Tri (hexyl, octyl, decyl)-citrat is regarded as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information