Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

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:

The study was conducted between 8 June 2009 and 16 July 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008 Date of Signature: 4/03/2009

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

EC Number:

935-853-6

IUPAC Name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Constituent 2

Reference substance name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

IUPAC Name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Analytical purity: 80 - 95%
- Lot/batch No.: 200901317-17
- Stability under test conditions : stable
- Storage condition of test material : approximately 4°C in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: obtained on 15 June 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK,
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: Not recorded
- Storage length: Not recorded.

- Preparation of inoculum for exposure:
The activated sewage sludge sample was washed three times by settlement and resuspension in culture 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 21ºC 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 deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC 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.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel. Rinsed three successive times with 10 ml of deionised reverse osmosis water.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 other: mg carbon/l

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture 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 approximately 21 °C, in darkness.

Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 24.3 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.

The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute 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 de-gassed water.


SAMPLING
-CO2 analysis:
Samples (2 ml) were taken from the control, standard and test material first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 12, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10, 12 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, standard and test material and on Day 0 for the toxicity control. All samples were analysed for CO2 immediately.

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 analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.


Dissolved Organic carbon (DOC) analysis:
Samples (20 ml) were removed from all culture vessels on Day 0 and from the control, standard and test material culture vessels on Day 28 and centrifuged (3,500 rpm, 15 minutes) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (27, 13 or 50 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.


CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An aliquot (133 ml) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 13.3 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb to filter matrices or to activated sewage sludge. However, 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 material in order to maintain consistency with a concurrent study.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 37.74 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test material 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

Parameter:

% degradation (CO2 evolution)

Value:

16

Sampling time:

28 d

Details on results:

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 material.

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 control Replicates R1 and R2, standard material Replicate R2 and test material Replicates 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 material attained 16% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC) gave percentage degradation values of 45% for both test material Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of test material into the microbial biomass prior to degradation, and hence CO2 evolution occurring.

The test material attained 16% degradation 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 52% degradation after 14 days and thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 112% degradation after 14 days and 118% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to an increase in the numbers of viable micro-organisms in the standard material vessels as a result of the readily biodegradable nature of the sodium benzoate. This effect occurs due to the micro-organisms utilizing the sodium benzoate as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100%.

Observations made throughout the test period showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved standard material or test material visible.

Any other information on results incl. tables

Percentage Biodegradation value:

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
2	38	1	19
6	55	0	34
8	55	0	35
10	96	0	45
12	98	0	67
14	112	0	52
21	103	5	-
28	109	5	-
29*	118	16	-

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

- = No degradation result obtained due to the toxicity control being terminated after 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 16% mineralisation after 28 days based on CO2 evolution and is therefore not considered as readily biodegradable under the strict terms and conditions of the OECD Guideline No. 301B.

Executive summary:

Introduction.A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in 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 OPPTS 835.3110 (Paragraph (m)).

Methods.The test material, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Conclusion.

The test material attained 16% mineralisation after 28 days based on CO2 evolution and is therefore not considered as readily biodegradable under the strict terms and conditions of the OECD Guideline No. 301B.