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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:
key study
Study period:
12 Feb 2010 - 19 Mar 2010
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)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Municipal activated sludge from the wastewater treatment plant, Mannheim, Germany.

Conditioning:
The inoculum was aerated in the laboratory until use. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh (mesh size approx 1mm). To reduce the influence of content of inorganic carbon in the blank controls, the activated sludge was aerated with carbon dioxude free air for approx. 48 hours at 22C.
On the day of exposure, the suspsension was washed with tap water, the aeration was stopped and the sludge allowed to settle. After setting, the supernatant was discarded and the remaining sludge suspension was filled up with tap water and the concentration of the sludge adjusted to 6.0g/L dry weight.

Aliquots of 7.5ml were added to the test vessels to obtain a sludge concentration of 30mg/L.
Duration of test (contact time):
28 d
Initial conc.:
25.5 mg/L
Based on:
test mat.
Remarks:
Corresponding to 20mg TOC/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST TEMPERATURE: 22C +/- 2C

TEST WATER:
Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
b) 36.4 g CaCl2 x 2 H2O filled up with pure water to 1000 mL volume
b) 22.5 g MgSO4 x 7 H2O filled up with pure water to 1000 mL volume
d) 0.25 g FeCl3 x 6 H2O filled up with pure water to 1000 mL volume
These solutions were used in the following amounts for the preparation of the test assays: 15ml of solution (a), 1.5ml of solution (b), 1.5ml of solution (c) and 1.5ml of solution (d).

TEST ITEM PREPARATION:
191mg of the test substance was weighed into a 25ml volumetric flask, and dissolved in acetone up to the mark. The TOC content of the test substance was 787mg/g. Aliquots of 5mls were dosed to the test vessels of the test substance and inhibition control. The organic solvent was removed by evaporation with compressed air before adding the distilled water. The same aliquots of organic solvent were added to the blank controls and then removed in the same way as the vessels containing the test substance.

REFERENCE ITEM PREPARATION:
A stock solution of 405.2mg/L aniline was prepared. This reference substance was dissolved in demineralised water and the contect of Total organic carbon (TOC) was calculated. Suitable aliquots (based on the calculation of the TOC content) were added to the test flask of the reference substance assay and the inhibition control assay (at 20mg/l TOC)

The following test assays were prepare
Solvent Red 500 (Test substance + inoculum) (2 samples: TS1 and TS 2)
CONTROLS:
Blank control: (inoculum only) (2 samples: BC1 and BC2)
Reference substance control: aniline reference item + inoculum
Inhibition Control: Test substance + Reference item + inoculum
The exact preparations are given in Table 1.

CARBON DIOXIDE EVOLUTION TESTS: DETAILS OF SAMPLE PREPARATION
The tests were performed in 2L bottles filled to 1.5L (see table 1 for details of all samples).

Each sample was prepared by as follows:

First 5ml aliquots of the test substance stock solution were added by pipette. (test substance and inhibition control)
The same volume of organic solvent only was added to the blanks
The solvent was removed by evaporation.
The required volumes of demineralised water were doesed to the vessles, and the aliquot of reference stock solution (reference and inhibition control).
The solutions of mineral salts were added and the pH adjusted.
The stock solution of activated sludge was added (equivalent to 30mg/L dry weight)
Full sample details are given in Table 1.

The bottles were connected to 2 serial scrubbing bottles (total volume 250ml) filled with 100ml 0.5M NaOH for the adsorption of CO2 from the biodegradation process. Twice a week the TIC values of the adsorption solutions of the first trap were determined and used for the calculation of produced CO2. After each sampling the second trap was moved forward and a new trap with fresh NaOH solution was placed in the second position. Each trap was analysed separately. The TIC of the NaOH solution was determined and considred by the calculation of biogenic produced CO2 amount. The incubation bottles were magnetically stirred. The aeration was performed with CO2 free air at a flow of approx 800mL/hour.

At the end of exposure, the pH values were measured in each test vessel. For stripping of
carbon dioxide, dissolved in the test medium, each test vessel was acidified by adding 2 mL
of concentrated hydrochloric acid. However, since the test substance was insufficiently
soluble in water, no useful measurements could be recorded from the inhibition control and
test substance assays. The aeration was continued for about 24 hours and the released
carbon dioxide amounts in both traps of each test vessel were determined and added to the
calculated amount of day 28.
Reference substance:
aniline
Key result
Parameter:
% degradation (CO2 evolution)
Value:
< 10
Sampling time:
28 d
Details on results:
The production of carbon dioxide (CO2) is a clear indication of biodegradation. The measured
amount of carbon dioxide at the end of the test is compared with the calculated maximal
theoretical production (ThCO2) and indicated as biodegradation degree in percent.
CO2 to ThCO2 is evaluated as follows:
60% within a 10 days window:readily biodegradable

Solvent Red 500 never reached a biodegradation of 10% or higher during the incubation time of 28 days. The mean biodegradation at test end after 28 days was <10% CO2/ThCO2.

The reference item aniline was sufficiently degraded to 85% after 14 days and to 91% after 28 days of incubation, thus confirming the suitability of
the aerobic activated sludge inoculum used.

In the inhibition control containing both, the test item and the reference item aniline, 36% biodegradation was noted within 14 days and 44% after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The biodegradation degree of Solvent Red 500 was <10% CO2/ThCO2 after a test duration of 28 days (mean from 2 test assays). Therefore Solvent Red 500 is considered to be not readily biodegradable under the conditions of this test.

The reference item aniline was sufficiently degraded to 85% after 14 days and to 91% after 28 days of incubation, thus confirming the suitability of
the aerobic activated sludge inoculum used.

In the inhibition control containing both, the test item and the reference item aniline, 36% biodegradation was noted within 14 days and 44% after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
Executive summary:

The criterion for ready biodegradability are as follows: the degradation of the test item of at least 60%, reached within a 10-day window;

Solvent Red 500 never reached a biodegradation of 10% or higher during the incubation time of 28 days.

Therefore, Solvent Red 500 is considered to be not readily biodegradable based on %CO2/ThCO2

The reference item aniline was sufficiently degraded to 85% after 14 days and to 91% after 28 days of incubation, thus confirming the suitability of

the aerobic activated sludge inoculum used.

In the inhibition control containing both, the test item and the reference item aniline, 36% biodegradation was noted within 14 days and 44% after 28 days of incubation.  According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Description of key information

The biodegradation degree of Solvent Red 500 was <10% CO2/ThCO2 after a test duration of 28 days (mean from 2 test assays).  Therefore Solvent Red 500 is considered to be not readily biodegradable under the conditions of this test.

Key value for chemical safety assessment

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

Additional information

The biodegradation degree of Solvent Red 500 was <10% CO2/ThCO2 after a test duration of 28 days (mean from 2 test assays).  Therefore Solvent Red 500 is considered to be not readily biodegradable under the conditions of this test.