Registration Dossier

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:
Between 30 March 2010 and 28 April 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :Lanolin Fatty Acids
Description : brown waxy solid
Batch number : 0000371886
Date received : 8 February 2010
Expiry date : 21 September 2009
Storage conditions :room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): obtained on
29 March 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture: not applicable
- Method of cultivation:not applicable

- Storage conditions: not stated in report
- Storage length: not stated in report

- Preparation of inoculum for exposure
The activated sewage sludge sample was washed three times by settlement and resuspension in culture 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 21ºC 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 105ºC 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 concentrationopen allclose all
Initial conc.:
13.1 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l

pH = 7.4

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 litre (final volume) of purified water* was 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

- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): not applicable
- Test temperature: 21°C
- pH: The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
- pH adjusted: not stated in report
- CEC (meq/100 g): not stated in report
- Aeration of dilution water: not stated in report
- Suspended solids concentration: not stated in report
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre glass culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
- Method used to create anaerobic conditions: not applicable
- Measuring equipment:
CO2 analysis: The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser.
TOC analysis: The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: 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 de-gassed water.
- Other:

SAMPLING
- Sampling frequency: Days 2, 6, 8, 10, 14, 21, 28 and 29
- Sampling method: not stated in report
- Sterility check if applicable: not applicable
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Toxicity control: For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (39.3 mg) was adhered onto a glass microscope slide* prior to addition to inoculated culture medium. An aliquot (51.4 ml) of the 1000 mg/l sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 13.1 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

STATISTICAL METHODS: No statistics reported
Reference substance
Reference substance:
other: Sodium benzoate

Results and discussion

Preliminary study:
No preliminary study described in report
Test performance:
The total CO2 evolution in the control vessels on Day 28 was 31.33 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
71
Sampling time:
28 d
Details on results:
The test item attained 71% degradation 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% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item has exhibited the potential for rapid degradation.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 94% degradation after 14 days and 112% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Any other information on results incl. tables

Following the recommendations of the International Standards Organisation (ISO 1995) the test item was adheared onto a glass microscope slide prior to addition to the test medium in order to aid dispersion of the test item in the test medium.

The IC content of the test item suspension in the mineraldium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to thethods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2present in the test vessels. Therefore any additional CO2detected in the Day 29 samples originated from dissolved CO2that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of control replicates R1and R2. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2into the second absorber vessels occurred.

The toxicity control attained 45% degradation after 14 days and thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. 

Analysis of the testdia taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), gave percentage degradation values of 100% and 98% respectively for Replicates R1and R2The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2evolution occurring.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test item attained 71% degradation 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% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item is a UVCB and has exhibited the potential for rapid degradation.
Executive summary:

Introduction. A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

Methods. The test item, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) the test item was adheared onto a glass microscope slide prior to addition to the test medium in order to aid dispersion of the test item in the test medium.

The degradation 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.

Results. The test item attained 71% degradation 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% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item is a UVCB and has exhibited the potential for rapid degradation.