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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
Study period:
2017
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)
Deviations:
yes
Remarks:
Minor deviation:On Day -1 of the study the temperature in a vessel containing water which was incubated under the same conditions as the test vessels was recorded as being 19.4 °C.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Hydroxypropyl, 2-, trimethylammonium formate
Physical state/Appearance: colorless to very light blue liquid
Batch: Chernil.20160628.B
CAS#: 62314-25-4
Purity: 95.25% active in water
Expiry Date: 28 June 2017
Storage Conditions: room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 12 December 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Duration of test (contact time):
> 0 - 28 d
Initial conc.:
10 mg/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The test item was dissolved directly in mineral medium.
A nominal amount of test item (300 mg) was dissolved in mineral medium and the volume adjusted to 1 liter to give a 300 mg/L stock solution. An aliquot (194 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 liters to
give a final concentration of 19.4 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
A nominal amount of test item (300 mg) was dissolved in mineral medium (1 liter) to give a 300 mg/L stock solution. Two samples were taken for DOC analysis; three untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to precondition the filter). A further nominal amount of test item (300 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. Two samples were taken for DOC analysis; one after
filtration through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to precondition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level
of 30 mg ss/L and then filtering or centrifuging as per the test item samples.
Test performance:
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 temperatures of between 19 and 24 °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 30 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 Hach HQ40d Flexi handheld 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 CO2-free air was produced by passing 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 CO2 absorbing solutions were prepared using purified water.


Key result
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
28 d
Details on results:
The test item attained 72% biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion,
whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, the test item has exhibited the potential for rapid biodegradation.

The toxicity control attained 60% biodegradation after 10 days, 66% biodegradation after 15 days and 65% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The
slight decrease in biodegradation between Days 10 and 28 was considered to be due to sampling/analytical variation.
Results with reference substance:
Sodium benzoate attained 82% biodegradation after 10 days, 76% biodegradation after 15 days and 88% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in biodegradation between Days 10 and 15
was considered to be due to sampling/analytical variation.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test item attained 72% biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion,
whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, the test item has exhibited the potential for rapid biodegradation.

Description of key information

The test item attained 72% biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, the test item has exhibited the potential for rapid biodegradation.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:
freshwater

Additional information