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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:
28 Jun 2017 to 30 Jan 2018
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives
Batch: P718261998
Purity: 97.7%
Physical state/Appearance: Brown liquid
Expiry Date: 01 June 2018
Storage Conditions: Room temperature 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 09 October 2017 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 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. 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 °C 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 3.4 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
14.9 mg/L
Based on:
test mat.
Remarks:
Equivalent to 10 mg carbon/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Mineral medium:
The mineral medium used in this study was that recommended in the OECD Guidelines. The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Preliminary solubility work:
The test item was insoluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) Handley et al (2002) it was concluded that the best testable dispersion was obtained when using the high shear mixing method of preparation.

Test Item Preparation:
The test item was dissolved directly in mineral medium. An amount of test item (44.7 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 litres to give a final concentration of 14.9 mg/L, equivalent to 10 mg carbon/L as recommended in the test guidelines.

Reference Item Preparation:
An initial stock solution of 1000 mg/L was prepared by dissolving the reference item (sodium benzoate) 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 litres 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. An amount of test item (44.7 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 14.9 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 litre test culture vessels each containing 3 litres 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).

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 between 22 to 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 26.5 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. If necessary 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
Preliminary study:
NA
Test performance:
The following validation criteria (as per the OECD Test Guidelines) were met, thus the study was considered valid.

The total CO2 evolution in the inoculum control vessels on Day 28 was 33.80 mg/L.

The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20%.

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 R1, test vessel R1 and R2, and the toxicity control. 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.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
14
Sampling time:
29 d
Details on results:
The test item attained 14% 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 47% biodegradation after 14 days and 40% 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. The slight decrease in biodegradation between Days 14 and 28 was considered to be due to sampling/analytical variation.

Sodium benzoate attained 69% biodegradation after 14 days and 69% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%, thereby confirming the suitability of the inoculum and test conditions.

Percentage Biodegradation Values

 

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

23

3

23

6

37

23

45

8

70

40

47

10

83

45

38

14

69

19

47

21

78

30

44

28

78

21

50

29*

69

14

40

* = Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives attained 14% 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:

An experimental study was conducted investigating the ready biodegradability of the test item, reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives, according to the OECD 301B Guideline (“Ready biodegradability, CO2 Evolution Test”) at a GLP accredited laboratory. The study satisfied all validation criteria given in the OECD Test Guidelines and is therefore considered valid and reliable.

 

The test item, at an initial concentration of 14.9 mg/L (equivalent to 10 mg carbon/L), was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 22 and 24 °C for 28 days. Biodegradation was assessed via determination of CO2 produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test item showed 14% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the OECD 301B Guideline.

Description of key information

An experimental study was conducted investigating the ready biodegradability of the test item, reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives, according to the OECD 301B Guideline (“Ready biodegradability, CO2 Evolution Test”) at a GLP accredited laboratory. The study satisfied all validation criteria given in the OECD Test Guidelines and is therefore considered valid and reliable.

 

The test item, at an initial concentration of 14.9 mg/L (equivalent to 10 mg carbon/L), was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 22 and 24 °C for 28 days. Biodegradation was assessed via determination of CO2 produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test item showed 14% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the OECD 301B Guideline.

Key value for chemical safety assessment

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

Additional information

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