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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:
13 June - 2 August 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:
Temperature was outside of limit of 22+/-2C in 4 days, but it is considered to have not affected the integrity or validity of the study
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
But they have not affected the integrity or validity of the study
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Remarks:
But they have not affected the integrity or validity of the study
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Expiry Date: 26 October 2017
Storage conditions: approximately 4C, in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 12 June 2017 for the initial experiment and the 3 July 2017 for the definitive test 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 twice by settlement and re-suspension in mineral medium to remove any excessive amounts of DOC that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of app. 21C and used on the day of collection. Suspended solids were determined by filtering of the washed activated sewage sludge using a Buchner funnel. Filtration then continued for 3 more minutes after rinsing the filter three successive times. The filter was dried at app. 105C for min 1 hour and was weighed after it cooled down.

Suspended solids concentration for the definitive test was 2.6 g/l prior to use.
Details on study design:
The following test preparations were prepared and inoculated in 5 L test culture vessels, each containing 3 L 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 C/L.
c) The test item in duplicate, in inoculated mineral medium to give a final concentration of 10 mg C/L.
d) The test item plus the reference in inoculated mineral medium to give a final concentration of 20 mg C/L to act as a toxicity control (single vessel).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids / L. The test was carried out in a temperature controlled room at temperatures of between 22 and 25C, in darkness. The pH was maintained at 7.4+/-0.2.

The following assessments were carried out:
- Observations
- pH measurements
- IC analysis
- IC/TC ratio

During Data Evaluation the following were calculated:
- Carbon content; for a 10 mg C/L content in a 3 L vessel was 30 mg/L for both the test and the reference items. Test item contains 60.29% carbon (per Sponsor data) and the reference item 58.34%.
- Percentage biodegradation (% ThCO2) was calculatd by substituting the IC values into the following equation: %ThCO2 = 100 x (mg IC in test flask - mg IC in control flask) / (mg TOC added as test chemical)
- Total CO2 Evolution = mf IC in control x (100 / %C of CO2) x (1 / test volume) = mg IC in control x (100 / 27.29) x (1 / 3)
Reference substance:
benzoic acid, sodium salt
Test performance:
- Total CO2 evolution in the inoculum control vessels on Day 28 was 34.07 mg/l and satisfied the relevant validation criterion given in the OECD Test Guidelines.
- The IC content of the test item suspension in the mineral medium at the start of the test was < 5% of the TC content and satisfied the relevant validation criterion given in the OECD Test Guidelines.
- Acidification of the test vessels on Day 28 was carried out in order to detect any dissolved CO2 that was created by the micro-organisms until Day 28.
- The results of the IC analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of Inoculum control Replicate 2 and Test Item Replicates 1 and 2. This decrease was considered to be due to sampling / analytical variation.
- IC 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.
- Biodegradation values in excess of 100% were considered to be due to an increase in the numbers of viable micro-organisms in the test item vessels as a result of the readily biodegradable nature of the test item. This is due to the micro-organisms utilising the test item as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100%.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 19
Sampling time:
28 d
Details on results:
- The test item attained 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Results with reference substance:
- Sodium benzoate attained 77% degradation after 14 days and 90% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
- The toxicity control attained 37% biodegradation after 14 days and 52% 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.

For the full results in tabular form and the degradation curves, please refer to the attached document CQ_Biodeg_tables_and_figures.pdf.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Executive summary:

Introduction

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)).

Methods

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 and 25C 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.

Results

The test item attained 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

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 and 25C 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 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information