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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
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)
Specific details on test material used for the study:
Physical state/Appearance:
brown opaque viscous liquid
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
-Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 6 March 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Sewage sludge/treatment micro-organisms were selected following the recommendations of guidelines. Pre-adaption of the inoculum was not performed.

-Preparation of Inoculum
The activated sewage sludge sample was washed twice by settlement and re-suspension 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 ºC and used on the day of collection. The suspended solids concentration was equal to 3.0 g/L prior to use.
Duration of test (contact time):
35 d
Initial conc.:
13.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of Test System
Based upon the results of the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995), the test item was dispersed directly on filter paper prior to dispersion in mineral medium.
An amount of test item (41.7 mg) was adsorbed onto a filter paper*, added to inoculated mineral medium and the volume was adjusted to 3 liters to give a final concentration of 13.9 mg/L, equivalent to 10 mg carbon/L.
A filter paper was added to each control vessel in order to maintain consistency between the test and procedure control vessels.

The following test preparations were prepared and inoculated in 5 liter glass test culture vessels each containing 3 liters of solution:
a) Duplicate inoculated control vessels consisting of inoculated mineral medium plus a filter paper.
b) Duplicate procedure control vessels containing the reference item (sodium benzoate) in inoculated mineral medium plus a filter paper* to give a final concentration of 10 mg carbon/L.
c) Duplicate test item vessels containing test item on a filter paper* in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) A single toxicity control consisting of the test item on a filter paper* 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).

A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

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, in darkness, and the temperature of the additional vessel containing water which was incubated under the same conditions as the test vessels ranged from 20 to 24 °C.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight.

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 CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
31
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
32
Sampling time:
36 d
Details on results:
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 36 showed an increase in all replicate vessels.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 36 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test item attained 31% biodegradation after 28 days and 32% biodegradation after 35 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 62% biodegradation after 14 days, 57% biodegradation after 28 days and 71% biodegradation after 35 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

The reference item, sodium benzoate, attained 82% biodegradation after 14 days, 78% biodegradation after 28 days, and 77% biodegradation after 35 days, thereby confirming the suitability of the inoculum and test conditions. The slight decrease in biodegradation between Days 28 and 35 was attributed to sampling/analytical variation.

       % Biodegradation
 Day  Procedure Control  Test Item  Toxicity Control
 0 0*   0  0
 2 49   0*  28
 6  71  0*  37
 8  80  4  47
 10  73  4  46
 14  82  6  62
 21  82  26  65
 28  78  31  57
 35  76  28  67
 36**  77  32  71

*Negative biodegradation values reported as 0% biodegradation

** Day 36 values were corrected to include carry-over of CO2 detected in absorber 2, if any

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test material attained 31% biodegradation after 28 days and 32% biodegradation after 35 days.
Executive summary:

The test material, attained 31% biodegradation after 28 days and 32% biodegradation after 35 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

The test material, attained 31% biodegradation after 28 days and 32% biodegradation after 35 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Additional information