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Environmental fate & pathways

Biodegradation in water: screening tests

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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 01 November 2016 and 29 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG301B in compliance with GLP, without deviations that influence the quality of the results

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
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
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
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EDA-BADGE-EDA
Cas Number:
854009-15-7
Molecular formula:
C25H40N4O4
IUPAC Name:
EDA-BADGE-EDA
Constituent 2
Chemical structure
Reference substance name:
EDA-BADGE-EDA-BADGE-EDA
Molecular formula:
C48H72N6O8
IUPAC Name:
EDA-BADGE-EDA-BADGE-EDA
Constituent 3
Chemical structure
Reference substance name:
EDA-BADGE α-glycol
Molecular formula:
C23H34N2O5
IUPAC Name:
EDA-BADGE α-glycol
Constituent 4
Reference substance name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Constituent 5
Reference substance name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Test material form:
liquid: viscous
Specific details on test material used for the study:
Identification: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine
Physical state/Appearance: yellow/ brown extremely viscous liquid
Batch: BBF01102V1
Purity: not supplied
Expiry Date: 01 January 2021
Storage Conditions: room temperature in the dark
Intended use/Application: substance to be used in industry

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 31 October 2016 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 once 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 3.7 g/L prior to use.
Duration of test (contact time):
ca. 28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 15.5 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:
Preliminary Solubility Work
The following preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation:
i) Ultrasonication and High Shear Mixing: A nominal amount of test item (300 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 an extremely cloudy homogenous dispersion. This was then subjected to high shear mixing (approximately 7500 rpm, 30 minutes) and formed a cloudy white dispersion with particles of test item visible dispersed throughout with a layer of foam on the surface.
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.
ii) Ultrasonication: A nominal amount of test item (50 mg) was dispersed in approximately 400 mL of mineral media with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a cloudy homogenous dispersion.
iii) High Shear Mixing: A nominal amount of test item (50 mg) was dispersed in approximately 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 a layer of foam and small particles visible on the surface.
iv) Adsorption onto an Inert Support: A nominal amount of test item (50 mg) was weighed on a filter paper. The filter paper was added to 3 liters of mineral media. This formed a clear colorless media column with test item adhered to the filter paper.
v) Addition onto an Inert Support: A nominal amount of test item (50 mg) was weighed on a glass slide. The glass slide was added to 3 liters of mineral media. This formed a clear colorless media column with test item adhered to the glass slide.
vi) Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fibre filter paper was attempted. The test item was insoluble in the following solvents dimethylformamide, tetrahydrofuran, acetone and chloroform. Therefore adsorption to filter paper in a volatile solvent was not possible.
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion was found to be obtained when using the ultrasonication method of preparation.

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 amount of test item (46.5 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (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 liters to give a final concentration of 15.5 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 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 24.3 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
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
All samples were analyzed for IC immediately. The remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required.
On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analyzed for IC using a Shimadzu TOC-LCSH TOC analyzer. Samples (135 µL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate


IC/TC Ratio
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.
IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 µL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680”C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.






Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 36.33 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. 0
Sampling time:
28 d
Remarks on result:
not determinable because of methodological limitations
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 R1, procedure control R1 and R2. 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.
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.
Therefore it was recommended, following the recommendations of the Test Guidelines that the test should be repeated at a reduced concentration of 5 mg C/L. However, due to the UVCB nature of the test item this was not performed at the request of the Sponsor.
Care should be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study.
The toxicity control attained 31% biodegradation after 14 days and 33% biodegradation after 28 days.
Sodium benzoate attained 78% biodegradation after 14 days and 77% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

Any other information on results incl. tables

Percentage Biodegradation Values:

 Day  % Bioodegradation      
   Procedure Control  Test Item  Toxicity Control
 0  0  0  0
 2  57  2  7
 6  59  0  35
 8  71  0  37
 10  71  0 31
 14  78  0 31
 21  79  0  25
 28  83  0  25
 29*  77 0  33

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
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 the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study
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; 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.

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 the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study.