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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-04 to 2016-03-11
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Isocarb 24
- Substance type: Product
- Lot/batch No.: ISC2403, 04058/MA
- Physical state: crystalline at ambient conditions

Study design

Oxygen conditions:
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):
35 d
Initial test substance concentrationopen allclose all
Initial conc.:
10.6 mg/L
Based on:
Initial conc.:
14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- 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 10 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 - 7.56
- 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.

- 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. On day 35, 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.

- 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)
- Method used to create aerobic conditions: The test system was aerated with CO2-free air at a rate of 30-100 mL/min, 1 functional control

- Sampling frequency: day 1, 4, 6, 8, 11, 14, 18, 21, 25, 28, 32, 35, 36 after acidification.

- Inoculum blank: yes (in duplicate)
- Toxicity control: yes
- Other: reference substance used was sodium benzoate (funcitional control)

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 ]

SigmaPlot (Windows), SPSS CORPORATION

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: mean of 4 of 6 replicates
Key result
% degradation (CO2 evolution)
ca. 52
Sampling time:
28 d
Remarks on result:
other: mean of all 6 replicates
Key result
% degradation (CO2 evolution)
> 60
Sampling time:
35 d
Remarks on result:
other: mean of all 6 replicates

BOD5 / COD results

Results with reference substance:
The percentage degradation of sodium benzoate reached the pass level of 60% within 6 days (66 %) and a maximum biodegradation of 100 % after 25 days.

Any other information on results incl. tables

In the toxicity control containing both test and reference item a biodegradation of 50 % was determined within 14 days and it came to 68% after 28 days and 78% after 35 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Applicant's summary and conclusion

Validity criteria fulfilled:
The differences of extremes of replicate values of removal of the test item at the end of the test was less than 20 %. The differences between replicates rely on bioavailability, four of the six replicates are clearly within 20 % from each other.
Interpretation of results:
readily biodegradable, but failing 10-day window
Under the test conditions the test item 2-decyltetradecanoic acid is classified as readily biodegradable, but failing the 10-day-window.
Executive summary:

The ready biodegradability of the test item 2 -decyltetradecanoic acid was determined with a non-adapted activated sludge over a test period of 35 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 10.6 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 35 by acidification of the test solutions. The last titration was made on day 36 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 100% after 25 days. In the toxicity control containing both test and reference item a biodegradation of 50 % was determined within 14 days and it came to 68% after 28 days and 78% after 35 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 35. The mean biodegradation on day 28 was 52 % and at test end on day 35 63%. Due to the difficult nature of the test item regarding bioavailability, six replicates with identical pre-treatment were used. Four replicates reached a mean of 63 % biodegradation after 28 days, therefore are considered to be readily biodegradable, but failing 10-day window without complying to the 10-day-window. Two replicates were 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.