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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:
Experimental start date 04 July 2017. Experimental completion date 04 October 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:
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)
Specific details on test material used for the study:
Identification: FSM-005W
CAS Number: 2601-33-4
Batch Number: 170321
Purity: 19.9% aqueous solution of FSM-005W (solid)
Physical state/Appearance: clear colorless liquid
Expiry Date: 20 November 2018
Storage Conditions: approximately 4 °C in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 04 September 2017 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 re-suspension 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* 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 3.0 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
69.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preliminary Work
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, the following work was conducted and samples analyzed for DOC using a Shimadzu TOC-VCPH TOC analyzer. During sample preparation, samples are either filtered or centrifuged to remove sewage sludge solids.
A nominal amount of test item (250 mg) was dissolved in mineral medium (500 mL) to give a 500 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further nominal amount of test item (250 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 500 mL. Two samples were taken for DOC analysis; one after filtration through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.

Test Item Preparation
The test item was dissolved directly in mineral medium.
A nominal amount of test item (1000 mg) was dissolved in mineral medium and the volume adjusted to 1 liter to give a 1000 mg/L stock solution. An aliquot (208.8 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 69.6 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.
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.

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 aliquot (208.8 mL) of the test item stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 liters to give a final concentration of 69.6 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.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item 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).
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
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 approximately 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 30.0 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. The pH was 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
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
Preliminary Investigational Work
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did adsorb to filter matrices but did not adsorb to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were centrifuged to remove the suspended solids present without the loss of any test item.

Validation Criteria and Biodegradation
The total CO2 evolution in the inoculum control vessels on Day 28 was 28.25 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 an increase in all replicate vessels with the exception of Procedure Control Replicate 2.
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 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
However care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study (10 mg carbon/L) as significant differences were shown between the Day 29 inoculum control and test item inorganic carbon values. Additionally the toxicity control vessel did not pass the validation criteria whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.
Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were statistically significant differences (P< 0.05) between the control and the test item. The test item was therefore considered to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
The toxicity control attained 6% biodegradation after 14 days and 7% biodegradation after 28 days thereby confirming that the test item did exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test at a concentration of 10 mg carbon/L.
Results with reference substance:
Sodium benzoate attained 71% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 77% biodegradation was attained. 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:
no
Remarks:
toxicity control vessel did not pass the validation criteria
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study (10 mg carbon/L) as significant differences were shown between the Day 29 inoculum control and test item inorganic carbon values. Additionally the toxicity control vessel did not pass the validation criteria whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.
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 approximately 22 and 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.

Results

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study (10 mg carbon/L) as significant differences were shown between the Day 29 inoculum control and test item inorganic carbon values. Additionally the toxicity control vessel did not pass the validation criteria whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.

Description of key information

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 approximately 22 and 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.

Results

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study (10 mg carbon/L) as significant differences were shown between the Day 29 inoculum control and test item inorganic carbon values. Additionally the toxicity control vessel did not pass the validation criteria whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.

Key value for chemical safety assessment

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

Additional information