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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-08 until 2015-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
OCSPP 835.3110 (Paragraph (m))
Deviations:
no
Principles of method if other than guideline:
not relevant
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Study design

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

PREPARATION OF INOCULUM
The activated sewage sludge 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 ºC 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 (rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven) 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 ºC 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 3.3 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
15.7 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of mineral medium according to OECD guideline:
Solution a: KH2PO4 8.50 g/L
(pH = 7.4) K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L

Solution b: CaCl2 27.50 g/L
Solution c: MgSO4.7H2O 22.50 g/L
Solution d: FeCl3.6H2O 0.25 g/L

To 1 liter (final volume) of reverse osmosis purified and deionized water were added the following volumes of solutions a – d:
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

- Temperature: 22 - 24 °C (temperature-controlled room)
- pH: 7.5 - 7.8 on Day 0
7.3 - 7.4 on Day 28
- pH adjusted: yes, if necessary to 7.4 +/- 0.2 using diluted hydrochloric acid or sodium hydroxide solution
- Suspended solids concentration in each vessel: 30 mg/L
- Continuous darkness: yes

TEST SETUP
Approximately 24 hours prior to addition of the test and reference items, the 5 liter test culture vessels were filled with 2400 mL of mineral medium and 27.3 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured.

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.

SAMPLING AND IC ANALYSIS
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 24, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. All samples were analyzed for inorganic carbon (IC) immediately, using a Tekmar-Dohrmann Apollo 9000 TOC analyzer or a Shimadzu TOC-V(CSH) TOC analyzer. Each analysis was carried out in triplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: activated sludge, no test item, no reference item
- Toxicity control: activated sludge, test item plus reference item
- Procedure control: activated sludge, no test item, with reference item
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The reference substance sodium benzoate attained 88% biodegradation after 14 days and 98% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The toxicity control attained 76% biodegradation after 14 days and 88% 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.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
20
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
64
Sampling time:
8 d
Parameter:
% degradation (CO2 evolution)
Value:
87
Sampling time:
21 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
93
Sampling time:
28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 93% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 to 24 °C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item attained 93% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.