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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 06 June 2017. Experimental completion date 04 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Version / remarks:

select appropriate one

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 13-0460
Chemical (IUPAC) Name: bis(cyclohexylmethyl) ether
CAS no: 14315-63-0
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 ºC

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 06 June 2017 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.0 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 liters) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.

Duration of test (contact time):

28 d

Initial conc.:

25 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper** Using filter paper the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
A nominal amount of test item (1072 mg) was dissolved in 10 mL of acetone to give a 1072 mg/10 mL solvent stock solution. An aliquot (25 μL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was added to inoculated mineral medium (107 mL) to give a final concentration of 25.0 mg/L, equivalent to 20 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.
A filter paper* was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (25 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was added to each vessel. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.
A test concentration of 20 mg carbon/L was employed in the study 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 (171.5 mL) of this stock solution was dispersed with inoculum (500 mL) and mineral medium to a final volume 5 liters, to give a test concentration of 34.3 mg/L, equivalent to 20 mg carbon/L. Aliquots (107 mL) of the 34.3 mg/L test concentration were dispensed to each of 33 replicate test vessels and the vessels sealed using Teflon lined silicon septa and aluminum crimp caps.
A filter paper*was added to the procedure control vessels in order to maintain consistency between the test and procedure control vessels.
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 study Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to 9 replicate test vessels.
An aliquot (25 μL) of the 1072 mg/10 mL test item solvent stock solution was dispensed separately on to 9 filter papers and the solvent allowed to evaporate to dryness for approximately 15 minutes.
The final concentration in the toxicity control vessels was 25.0 mg test item/L plus 34.3 mg reference item/L, equivalent to 40 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and incubated in 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution:
a) An inoculated control consisting of inoculated mineral medium, plus a filter paper*, 33 replicate vessels.
b) The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium, plus a filter paper*, to give a final concentration of 20 mg carbon/L, 33 replicate vessels.
c) The test item, on a filter paper*, in inoculated mineral medium to give a final concentration of 20 mg carbon/L, 29 replicate vessels.
d) The test item, on a filter paper*, plus the reference item in inoculated mineral medium to give a final concentration of 40 mg carbon/L to act as a toxicity control, 9 replicate vessels.
A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Test media a) to d) were inoculated with the prepared inoculum at a final concentration of 100 mL/L.
Aliquots (107 mL) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the inoculum control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional inoculum control and procedure control were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on Days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminum crimp caps and incubated in darkness at approximately 20 oC with constant shaking at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Details on results:

Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.

Results with reference substance:

DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 showed that the replicate reference item vessels attained 100% biodegradation for each replicate vessel. The biodegradation rates for the reference item were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to biodegradation and hence CO2 evolution occurring.

Validation Criteria

The mean TIC in the inoculum control vessels on Day 28 was 0.39 mg/L; equivalent to 2.0% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Sodium benzoate attained 82% biodegradation after 14 days and 72% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The decrease in the biodegradation values were considered to be due to sampling/analytical variation.

The toxicity control attained 42% biodegradation after 14 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Validity criteria fulfilled:

yes

Remarks:

Results with sodium benzoate confirmed the suitability of the inoculum and test conditions. The toxicity control showed test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Interpretation of results:

not readily biodegradable

Conclusions:

The substance showed 0% biodegradation in an OECD TG 310 test and is considered to be not biodegradable.

Executive summary:

The ready biodegradability of FRET 13 -0460 was investigated in a study conducted in accordance with OECD TG 310 (CO2 headspace test) and GLP. The concentration tested was 25 mg/l test substance. The test substance biodegrades for 0% and it was not toxic to the inoculum (82% biodegradation of the sodium benzonate reference substance)

Description of key information

The ready biodegradability of FRET 13 -0460 was investigated in a study conducted in accordance with OECD TG 310 (CO2 headspace test) and GLP. The concentration tested was 25 mg/l test substance. The test substance biodegrades for 0% and it was not toxic to the inoculum (82% biodegradation of the sodium benzonate reference substance)

.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater