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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
Specific details on test material used for the study:
Identification: RL 610/11
Description: clear colorless liquid
Batch: 1023M18305
Purity: 100%
Expiry / retest date: not supplied
Storage conditions: room temperature in the dark

Oxygen conditions:
Inoculum or test system:
sewage, domestic, adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 21 May 2012 from the aeration stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through preweighed GFIA filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.8 g/L prior to use.
Details on study design:
Mineral Medium
The mineral medium used in this study (see Appendix 2) was that recommended in the OECD Guidelines.

The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at approximately 21°C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 32.1 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter. If necessary the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer. The COrfree air was produced by £assing compressed air through a glass column containing self-indicating soda lime (Carbosorb ) granules.

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The C02 absorbing solutions were prepared using purified de-gassed water.

The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
From the pre-study solubility work it was concluded that the best methods of preparation were by either ultrasonication, high shear mixing, silica gel followed by high shear mixing or by preparing a preliminary solvent stock solution in acetone and adding an aliquot onto filter paper and then subjecting to high shear mixing. As there were no differences in the dispersibility of the test item between these methods, the test item was prepared using ultrasonication.
Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 31.54 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satified the validation creiterion given in the OECD guidelines.
Acidification of the test vessels on Day 28 followed by final analyses on Day 29 was conducted according to methods specified in the test guidelines. The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. Inorganic carbon analysis of the samples from the secon absorber vessels on Day 29 confirmed that mo significant carry-over of CO2 into the second absorver vessels occurred.
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: satisfied 10-day window
Results with reference substance:
The reference substance (sodium benzoate) attained 76% degradation after 14 days and 77% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Sodium benzoate attained 76% degradation after 14 days and 77% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The toxicity control attained 62% degradation after 14 days and 78% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test item attained 92% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions ofOECD Guideline No. 301B.

Description of key information

Clark (2012) provides a reliable study which showed test substance degraded 92% in 28 days and meeting the 10 day window criteria.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A reliable measured biodegradation study for FX511 is available and included in the dossier.


Clark (2012) conducted a reliable (Klimisch 1) GLP compliant study according to OECD 301 B (CO2 Evolution Test) method. The biodegradability of FX511 exposed to microorganisms derived from activated sludge obtained from a municipal wastewater treatment plant was investigated under aerobic exposure conditions. After 28 days the test substance showed 92% degradation, with an initial test concentration of 10 mgC/L. Greater than 60% degradation occurred within 10 day window. The study concluded that FX511 can be considered to be readily biodegradable, passing the 10-day window.Consequently this study will be taken for the biodegradation in water endpoint.