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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained on 28 June 2016 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preliminary Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was negilible. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO 10634, (1995)) and in the published literature (Handley et al, 2002) the test item was adhered onto a glass slide (76 x 26 mm) prior to dispersion in mineral medium to ensure that all the test item is added into the test vessels and that none is lost through adhering to glassware used to weigh the test item out in.

An amount of test item (58.2 mg) was adhered onto a glass slide (76 x 26 mm) prior to dispersal in the test vessel and the volume adjusted to 3 liters to give a final concentration of 19.4 mg/L, equivalent to 10 mg carbon/L.

A glass slide (76 x 26 mm) was added to each control vessel in order to maintain consistency between the test and procedure control vessels.

A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

A glass slide (76 x 26 mm) was added to each procedure control vessel in order to maintain
consistency between the test and procedure control vessels.

Toxicity Control
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 (58.2 mg) was adhered onto a glass slide (76 x 26 mm) prior to dispersal in the test vessel. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 19.4 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System
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 plus a glass slide (76 x 26 mm).
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a glass slide (76 x 26 mm) to give a final concentration of 10 mg carbon/L.
c) The test item on a glass slide (76 x 26 mm), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item on a glass slide 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).

A glass slide was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

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 temperatures of between 22 and 24 °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 22.5 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 Hach HQ40d Flexi handheld meter prior to the pH being 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 CO2-free air.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute 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.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
4
Sampling time:
28 d
Details on results:
Validation Criteria and Biodegradation
The total CO2 evolution in the inoculum control vessels on Day 28 was 39.40 mg/L and therefore satisfied the 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 (see Table 3) 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 item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of test item Replicate 1. This decrease was considered to be due to sampling/analytical variation.

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 item attained 4% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 33% biodegradation after 14 days and 45% 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.

Sodium benzoate attained 93% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 92% biodegradation was attained. The slight decrease in biodegradation between Days 14 and 28 was considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 4% 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 24 °C for 28 days.

An approach endorsed by the International Standards Organisation (ISO 10634, (1995)) and the published literature (Handley et al, 2002) is to adsorb the test item onto an inert support

prior to dispersion in the test vessels.  Using this method ensures that all the test item is added into the test vessels and that none is lost through adhering to glassware used to weigh the test

item out in.

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 4% 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

The test item attained 4% 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