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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-30 until 2017-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Date of Inspection, 29.06.2016

Test material

Constituent 1
Chemical structure
Reference substance name:
Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-,C12-13-branched alkyl esters
EC Number:
947-004-7
Molecular formula:
C28H56O6 - C29H58O6 - C30H60O6
IUPAC Name:
Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-,C12-13-branched alkyl esters
Test material form:
liquid
Details on test material:
product

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal wastewater treatment plant Breisgauer Bucht, Freiburg, Germany
- Sampling date: December 05th, 2016
- Storage conditions: not applicable
- Storage length: immediately used
- Preparation of inoculum for exposure: Washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in tap water. It was aerated with CO2-free air overnight.
Duration of test (contact time):
35 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
other: organic carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Mineral Medium
A: Potassium dihydrogenphosphate KH2PO4 8.50 g, Dipotassium hydrogenphosphate K2HPO4 21.75 g, Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g, Ammonium chloride NH4Cl 0.50 g, are dissolved in demineralised
water and made up to 1 litre.
B: Calcium chloride dihydrate CaCl2 * 2H2O 36.4 g, is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g, is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g, is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre. For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions
(B), (C) and (D) are added and the volume is made up to 1 litre.
CO2-absorption medium: 72.07 g NaOH was dissolved in 9 L deionised water in closed vessels (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined.
- Continuous darkness: no - diffuse light for 35 days

TEST SYSTEM
- Culturing apparatus: The CO2-free air production system consists of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.1 M NaOH (sodium hydroxide). At the end of the system is one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A colour change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with two input and 22 output channels and through PE-tubes.
- Number of culture flasks/concentration: In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.
- Measuring equipment: IC measurement was performed with a total carbon analyser (TOC-5050A Shimadzu with an autosampler ASI-5000A) by purging the inorganic carbon with H3PO4 (25%) using a non dispersive infrared (NDIR) detector.
- Other: Because of its insolubility the test item was added directly into the test vessels. As carrier material silica gel was used. Approximately 500 mg of silica gel were weighed out in a mound in a small weighing boat. A spatula was used to tap the top of the mound and flatten it so that it became a “plateau”. 41.4 – 41.9 mg of the test material was added onto the silica gel. The weighing boat was then folded and the test material dispersed on silica gel was poured into the test vessels. This corresponds to a concentration of 19.2 – 19.5 mg/L organic carbon.


SAMPLING
- Sampling frequency: On the 3th, 7th, 10th, 14th, 21st, 28th, 35th day 4 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined.
- Other: The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. Measurement of the reference vessels was stopped on day 28, since the degradation had already reached the plateau phase. Therefore 2 mL of 4M hydrochloric acid (HCl) was added into the three reactors with the reference item to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line. Last measurement of the blanks, the test item and the toxicity control was performed on day 35. For that the IC's were determined in the first CO2-absorber flasks, and the reactors were acidified as described above. On the next day (day 36) the IC was determined in both CO2-absorber flasks in line.

CONTROL AND BLANK SYSTEM
- Inoculum blank: three reactors containing only inoculum
- Reference substance: A stock solution of 10 g/L sodium benzoate in water was prepared. 5.15 mL of this stock solution were added into the reference vessels corresponding to a concentration of 20 mg/L organic carbon.
- Toxicity control: 43.4 mg of the test item were added via silica gel directly into the toxicity control vessel. Additionally, 5.15 mL of the reference stock solution were added to the vessel. This corresponds to a concentration of 40.2 mg/L organic carbon.


STATISTICAL METHODS:
- Theoretical CO2 amount of the test item: ThCO2 [mg] = weight of added test item [mg] * carbon-content [mg/mg] * 44/12
- Amount of CO2 released from the reactors: CO2 [mg/1500 mL] = IC [mg/L]* Volumeabsorber flask [L] *44/12
- Amount of CO2 removed for IC-measurement: CO2 total (i, x) = CO2 absorber flask (i, x) + Sum CO2 sampling (i, x-1)
- Percentage biodegradation of the reference item: BiodegradationCO2 [%]=100*(CO2 Reference reactor [mg] - CO2 Blank reactor [mg])/ThCO2 [mg]
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
64.2
Sampling time:
35 d
Remarks on result:
other: after acidification, mean of the three replicates
Key result
Parameter:
% degradation (CO2 evolution)
Value:
54
Sampling time:
28 d
Remarks on result:
other: after acidification, mean of the three replicates
Details on results:
According to the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 the test item can be classified as inherently biodegradable. The reference compound sodium benzoate reached the pass levels for ready biodegradability within 7 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The degradation of the test item after 35 days was 64.2% (after acidification, mean of three replicates). The degradation after 28 days was 54.0%.
According to the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 the test item can be classified as inherently biodegradable. The degradation of the toxicity control after 14 days was 58.6%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.
Executive summary:

The biodegradability of Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-, C12-13-branched alkyl esters was assessed in a CO2 -evolution test according to the OECD 301B guideline (July 1992).

A solution of the test item in mineral medium corresponding to 20 mg TOC/L was inoculated with activated sludge (30 mg d.s./L).

The test item was added directly into the test vessels. Carrier material was silica gel.

The test vessels were aerated by the passage of carbon dioxide-free air and incubated under aerobic conditions in diffuse light for 35 days. Degradation was followed by determining the carbon dioxide produced and adsorbed to sodium hydroxide via IC-measurement (IC=inorganic carbon).

The temperature was 19.4 – 22.0°C throughout the whole study. The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 2.6 – 4.4 bubbles/second).

All validity criteria were fulfilled:

- The IC content in the test vessel was less than 5% of the TOC introduced with the test item.

- The CO2 evolution in the inoculum blank at the end of the test was below 40 mg/L.

- The difference of extremes of replicate values at the end of the 10-d window and at the end of the test was less than 20%.

- The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 7.

- The degradation extent in the toxicity control was above 25% in 14 days based on ThCO2.

The degradation extent of the test item was 64.2 % within 35 days after acidification.

The degradation after 28 days was 54.0%. According to the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 the test item can be classified as inherently biodegradable.

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