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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:
2016-02-04 to 2016-03-24
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: 2016-01-27
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 18.6 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.:
10.4 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): Emulsifier Alfoterra S23-11S 90 (Alcohols, C12-13-branched and linear, propoxylated, sulphated, sodium salts (11 PO)), Batch No. 12/006/S51, Received 2015-10-23, Expiry date 2017-10-23 (acc. to test facility SOP), Stock solution 5 g/L in ultrapure water, Final concentration 5 mg/L in the test vessel,
Additionally Silicagel was used (type and concentration if used): SIGMA ALDRICH, high-purity grade (Merck Grade 9385) pore size 60 A, 230-400 mesh particle size, Batch No. BCBP2293V, CAS-No 112926-00-8, Received 2015-06-15, Expiry date 2017-06-16 (acc. to test facility SOP); 500 mg/42 mg test item (weighed out with the test item in 20 mL GC-Vial), Final concentration 500 mg/3 L in the test vessel

- Test temperature: 22 ± 2 °C
- pH: Start: not mentioned; End prior to Acidification: Day 35: 7.47 - 7.50
- pH adjusted: no
- Continuous darkness: Low light conditions (brown glass bottles)
- Other: Dispersion treatment: Continuous stirring, Aeration 30 - 100 mL/min

Pretreatment: Test item weighed out with 500 mg silica gel in GC-Vial (20 mL). Heating to 120°C for at least ten minutes (until the test item is fully melt), then vortexing for at least one minute (repeated at least 3 times). This was prepared in advance and then stored until test start at room temperature in the closed vials.

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 (pre-treated as described above) 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. Appropriate amounts of emulsifier stock solution were added 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 48, 1 mL 37 % HCl was added to each of the vessels. 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.


- Number of culture flasks/concentration: 6 with test substance, 2 with silica gel control (Emulsifier and silica gel without test item 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. emulsifier and pre-treatment as described above) and reference item in test concentration), 1 functional 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, 21, 25, 28, 32, 35, 39, 42, 46, 48, 49 after acidification.

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

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 ]


Software Excel, MICROSOFT CORPORATION
SigmaPlot (Windows), SPSS CORPORATION


Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
65
Sampling time:
28 d
Remarks on result:
other: mean of 2 of 6 replicates
Parameter:
% degradation (CO2 evolution)
Value:
39
Sampling time:
28 d
Remarks on result:
other: mean of all 6 replicates
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
48 d
Remarks on result:
other: mean of all 6 replicates
Results with reference substance:
The percentage degradation of sodium benzoate reached the pass level of 60% within 8 days (67 %) and a maximum biodegradation of 85 % after 48 days.

In the toxicity control containing both test and reference item a biodegradation of 49 % was determined within 14 days and it came to 62% after 28 days and 74% after 48 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 of extremes of replicate values of removal of the test item after 28 days , as well as at the end of the test was less than 20 %. The differences in biodegradation can be assigned to the differences in bioavailability.
Interpretation of results:
readily biodegradable
Conclusions:
The test item 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-,tris(C14-C15-alkyl)esters is classified as readily biodegradable.

Executive summary:

The ready biodegradability of the test item 1,2,3 -Propanetricarboxylic acid, 2 -Hydroxy-, tris(C14 -C15 -alkyl)ester) was determined with a non-adapted activated sludge over a test period of 48 days in the Modified Sturm Test according to OECD 301 B. The test item was tested at a concentration of 14 mg/L with 6 replicates corresponding to a carbon content (TOC) of 10.4 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 48 by acidification of the test solutions. The last titration was made on day 49 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 8 days and a maximum biodegradation of 85% after 48 days. In the toxicity control containing both test and reference item a biodegradation of 49 % was determined within 14 days and it came to 62% after 28 days and 74% after 48 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) on day 11. The 60 % pass level was reached by the mean of replicates on day 42. The mean biodegradation on day 28 was 39% and at test end on day 48, 72%. Due to the difficult nature of the test item regarding bioavailability, six replicates with identical pre-treatment were used. Two replicates reached a mean of 65 % biodegradation after 28 days, therefore are considered to be readily biodegradable. Three replicates are lagging behind, but passing the 60 % level at least after 46 days. One replicate didn’t reach the 60% threshold until test end, but the biodegradation was still increasing. 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. The maximum of biodegradation reached by the 2 best biodegradable replicates was 84 % and 100 %, respectively. This indicates the potential for complete mineralization.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradability of the test item 1,2,3 -Propanetricarboxylic acid, 2 -Hydroxy-, tris(C14 -C15 -alkyl)ester) was determined with a non-adapted activated sludge over a test period of 48 days in the Modified Sturm Test according to OECD 301 B. The test item was tested at a concentration of 14 mg/L with 6 replicates corresponding to a carbon content (TOC) of 10.4 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 48 by acidification of the test solutions. The last titration was made on day 49 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 8 days and a maximum biodegradation of 85% after 48 days. In the toxicity control containing both test and reference item a biodegradation of 49 % was determined within 14 days and it came to 62% after 28 days and 74% after 48 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) on day 11. The 60 % pass level was reached by the mean of replicates on day 42. The mean biodegradation on day 28 was 39% and at test end on day 48, 72%. Due to the difficult nature of the test item regarding bioavailability, six replicates with identical pre-treatment were used. Two replicates reached a mean of 65 % biodegradation after 28 days, therefore are considered to be readily biodegradable. Three replicates are lagging behind, but passing the 60 % level at least after 46 days. One replicate didn’t reach the 60% threshold until test end, but the biodegradation was still increasing. 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. The maximum of biodegradation reached by the 2 best biodegradable replicates was 84 % and 100 %, respectively. This indicates the potential for complete mineralization. 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-,tris(C14-C15-alkyl)esters is classified as readily biodegradable.

Additionally data are available for a structurally related substance Tri (hexyl, octyl, decyl) citrate with the only difference in shorter carbon chain ester groups. The substance exhibited up to 67% biodegradation after 28 d in a ready biodegradability test. The biodegradation of the test substance reached 51% at the end of the 10-d window. But for UVCB substances the 10-day window condition should not be applied. In a study on inherent biodegradability the pass level of 70% was reached. The substance can be considered as readily biodegradable.