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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: 1 ,4-cyclohexanedicarboxylic acid
Description: white powder
Batch: TS100414
Purity: 99.39%
Storage Conditions: room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 28 November 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. The activated sewage sludge sample was washed once 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 pre-weighed GF/A 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 deionised 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 2.0 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
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 45.0 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 340I 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 C02 free air. The test vessels were sealed and C02-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer. The C02-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb ) granules. The C02 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. Samples (2 mL) were taken from the first C02 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29. The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analyzed for C02 immediately. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
103
Sampling time:
28 d
Details on results:
The test item attained 103% 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 of OECD Guideline No 301 B. Degradation values in excess of 100% degradation were considered to be due to sampling/analytical variation. The toxicity control attained 80% degradation after 14 days and 83% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained 87% degradation after 14 days and 90% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 87% degradation after 14 days and 90% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium following the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; C02 Evolution Test". The test item attained 103% 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 of OECD Guideline No 301B. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.