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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 07 March 2017 and 05 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
EC No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid: viscous
Details on test material:
Identification:
1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Appearance/Physical state:
clear, colorless, viscous liquid
Batch:
WA 1508
Purity:
100% UVCB
Expiry date:
01 January 2021
Storage conditions:
room temperature, in the dark
Specific details on test material used for the study:
Identification : 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Physical state/Appearance: clear colorless viscous liquid
Batch : WA 1508
Purity : not applicable – complex mixture
Expiry Date : 01 January 2021
Storage Conditions: room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on
6 March 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):
ca. 28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 17.1 mg/L
Based on:
test mat.
Initial conc.:
ca. 10 mg/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Experimental Design and Study Conduct

Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was partly soluble in water. Therefore preliminary solubility/dispersibility work was performed, as follows, in order to determine the most suitable method of preparation:

i) Ultrasonication: A nominal amount of test item (100 mg) was dispersed in 1 liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute prior to ultrasonication for 30 minutes. This formed a clear colorless water column with test item visible adhered to the bottom of the vessel.
ii) High Shear Mixing: A nominal amount of test item (100 mg) was dispersed in 1 liter of deionized reverse osmosis water with the aid of high shear mixing (approximately 7500 rpm, 30 minutes). This formed a hazy dispersion with particles of test item visible dispersed throughout and test item adhered to the vessel and mixer head.
This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.
iii) Ultrasonication: A nominal amount of test item (50 mg) was dispersed in 400 mL of mineral media with the aid of ultrasonication for 15 minutes. After sonication the test item adhered to the bottom of the vessel and could not be washed out.
iv) High Shear Mixing: A nominal amount of test item (50 mg) was dispersed in
400 mL of mineral media with the aid of high shear mixing (approximately
7500 rpm, 15 minutes). The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a hazy dispersion with particles of test item visible dispersed throughout and adhered to the vessel and high shear mixer head.
v) Adsorption onto an Inert Support: A nominal amount of test item (50 mg) was weighed onto 500 mg of silica gel. The silica gel was dispersed in 400 mL of mineral media with the aid of high shear mixing (approximately 7500 rpm,
15 minutes). The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a cloudy dispersion with particles of silica gel/test item visible dispersed throughout. After 72 hours of magnetic stirring the appearance remained unchanged.
vi) Adsorption onto an Inert Support: A nominal amount of test item (50 mg) was weighed onto a filter paper. The filter paper was added to 3 liters of mineral media. This formed a clear colorless media column with the test item visible adhered to the filter paper and at the bottom of the vessel. After 24 and 48 hours of magnetic stirring a cloudy dispersion was formed with an oily sheen of test item visible on the surface and broken up pieces of filter paper visible dispersed throughout.
vii) Addition onto an Inert Support: A nominal amount of test item (50 mg) was weighed onto a glass slide. The glass slide was added to 3 liters of mineral media. This formed a clear colorless media column with test item visible adhered to the glass slide at the bottom of the vessel. After 72 hours of magnetic stirring the appearance remained unchanged.
viii) Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fibre filter paper was attempted. A nominal amount of test item (1000 mg) was dissolved in acetone (10 mL) with the aid of shaking by hand for approximately 1 minute, and formed a clear colorless solution. An aliquot (450 µL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then added to 400 mL of mineral medium and subjected to high shear mixing (approximately 7500 rpm, 5 minutes). The volume was then adjusted to 3 liters with mineral medium. This formed a cloudy dispersion containing broken up pieces of filter paper throughout with an oily sheen of test item visible on the surface.
ix) Preliminary Solution in a Non-Volatile, Non-Degradable Solvent: A nominal amount of test item (100 mg) was dispersed in silicone oil (10 mL) with the aid of shaking by hand for 1 minute followed by ultrasonication for 17 minutes. A cloudy white dispersion was formed.
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, (1995)) it was concluded that the best testable dispersion was found to be obtained when using the silica gel 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 adsorbed onto silica gel prior to dispersion in mineral medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.
An amount of test item (41.7 mg) was adsorbed onto the surface of 500 mg of granular silica gel (230-400 mesh Sigma Lot No TBC3040V) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). The test item/silica gel/mineral mineral medium dispersion was then dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 13.9 mg/L,
Silica gel (500 mg) was added to each control vessel in order to maintain consistency between the test and procedure control vessels. The silica gel was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm,
15 minutes) prior to addition to each vessel.
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 with the aid of ultrasonication for approximately 10 minutes. 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.
Silica gel (500 mg) was also added to the procedure control vessels in order to maintain consistency between the test and procedure control vessels. The silica gel was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to addition to each vessel.

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 (41.7 mg) was adsorbed onto the surface of 500 mg of granular silica gel (230-400 mesh Sigma Lot No.TBC3040V) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). The test item/silica gel/mineral medium dispersion was then dispersed in inoculated mineral medium and an aliquot (51.4 mL) of the sodium benzoate stock solution added. The volume was adjusted to 3 liters to give a final concentration of 13.9 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 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus 500 mg silica gel.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Silica gel 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 23 and 25 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 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/min 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

Assessments
Observations
The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH Measurements
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

IC Analysis
See "Details on Analytical Methods" (above).
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 31.39 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 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.
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
28 d
Remarks on result:
other:
Remarks:
Not readily biodegradable
Details on results:
Biodegradation
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 inoculum control Replicate 1 and the toxicity control.
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 1% 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 38% biodegradation after 14 and 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 69% biodegradation after 14 days and 71% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Percentage Biodegradation Values:

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

48

0

28

6

74

0

40

8

72

0

36

10

69

2

44

14

69

0

38

21

70

0

41

28

67

0

42

29*

71

1

38


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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 1% 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:

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; CO2Evolution 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 23 and 25 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)) and the published literature (Handleyet al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

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 1% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.