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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:
from 2007-11-06 to 2007-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of activated sewage sludge microorganisms was obtained on 5 November 2007 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and re-suspension 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 21 °C and used on the day of collection.
- Pre-treatment: Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 mL of culture medium and 34.6 mL of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
- Concentration of sludge: 30 mg suspended solids/L
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper
Duration of test (contact time):
ca. 28 d
Initial conc.:
16.6 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
10 mL of solution a: KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4.2H2O 33.40 g/L; NH4CI 0.50 g/L; pH 7.4
1 mL of solution b: CaCl2 27.50 g/L
1 mL of solution c: MgSO4.7H20 22.50 g/L
1 mL of solution d: FeCl3.6H2O 0.25 g/L add to 1 litre (final volume) of purified water.
- Solubilising agent: Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Test temperature: 21 °C
- pH: 7.4
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg ss/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre glass vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/minute and stirred continuously by magnetic stirrer.
- Measuring equipment:

FOR CO2 ANALYSIS: The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

FOR DOC ANALYSIS: On Days 0 and 28 samples (20 mL) were removed from all culture vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

- Test performed in open system: no, vessels were sealed
- 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.

SAMPLING FOR CO2 ANLYSIS:
- Sampling frequency: Day 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29.
- Sampling method: Samples (2 mL) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.

SAMPLING FOR DISSOLVED ORGANIC CARBON (DOC) ANALYSIS:
- Samling frquency: On Days 0 and 28 samples (20 mL) were removed from all culture vessels and filtered through Gelman 0.45 urn Acrocap filters (approximately 5 mL discarded) prior to DOC analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes, 16.6 mg/L test material and 17.1 mg/L sodium benzoate (= 20 mg/L carbon)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb to filter matrices or to activated sewage sludge. For the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test material.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
> 78
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
> 60
Remarks on result:
other: 10 day window
Details on results:
The total CO2 evolution in the control vessels on Day 28 was 25.93 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test was below 5 % of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20 % 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 the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that 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 material.
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 replicate R2 and test material replicate R2. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test material attained 78 % 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 material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
The toxicity control attained 94 % degradation after 14 days and 84 % degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The decrease in degradation obtained for the toxicity control between Day 14 and Day 28 was considered to be due to a slightly greater increase in CO2 evolution values in the control inorganic carbon values compared to the increase in inorganic carbon within the toxicity control vessel during this time period.
Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC) gave percentage degradation values of 100 % for both the test material Replicates R1 and R2 and 100 % for the toxicity control. Sodium benzoate attained 100 % degradation for both Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of test material/sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.
Observations made throughout the test period showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels were light brown dispersions with no undissolved standard material visible. The test material vessels were observed to contain light brown dispersions with no undissolved test material visible. The toxicity control vessel contained a light brown dispersion with no undissolved test or standard material visible.
Results with reference substance:
Sodium benzoate attained 85 % degradation after 14 days and 88 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test material attained 78 % 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 material can, therefore, be considered as 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 material in an aerobic aqueous medium. The test followed that described method in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

The test material, 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. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes. The test material attained 78 % 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 material can, therefore, be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

The test material attained 78 % degradation after 28 days and satisfied the 10-Day window validation criterion as tested in a study according to OECD TG 301B. Therefore, the test material is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test followed that described method in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

The test material, 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. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes. The test material attained 78 % 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 material can, therefore, be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.