Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

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:

Experimental start date 05 September 2017 Experimental completion date 08 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

EC Number:

217-062-1

EC Name:

Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

Cas Number:

1732-96-3

Molecular formula:

C20H10O10

IUPAC Name:

2-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyloxy)ethyl 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxylate

impurity 1

Reference substance name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

EC Number:

209-008-0

EC Name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

Cas Number:

552-30-7

Molecular formula:

C9H4O5

IUPAC Name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

impurity 2

Reference substance name:

not assignable

IUPAC Name:

not assignable

Specific details on test material used for the study:

Identification: RIKACID TMEG-500
Batch: 5259
Purity: 81.1%
Physical state/Appearance: cream colored powder
Expiry Date: 24 April 2018
Storage Conditions: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 9 October 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 3.4 g/L prior to use.

*Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven

Duration of test (contact time):

27 d

Initial test substance concentration

Initial conc.:

22.7 mg/L

Based on:

test mat.

Remarks:

equivalent to 10 mg carbon/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item slowly hydrolyzed and dissolved in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
An initial test was terminated after 17 days due to a greater than 20% difference in the inorganic carbon values between the replicate test item vessels. Therefore the test was re-started.
The test item was dispersed directly in mineral medium.
An amount of test item (68.1 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 22.7 mg/L, equivalent to 10 mg carbon/L

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 (68.1 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 22.7 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 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. 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.

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.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Test performance:

The total CO2 evolution in the inoculum control vessels on Day 28 was 33.80 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

Parameter:

% degradation (CO2 evolution)

Value:

84

Sampling time:

28 d

Remarks on result:

other: satisfied the 10-Day window validation criterion

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 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 Replicate 1 and test item Replicate 1.
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 84% 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%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 70% biodegradation after 14 days and 72% 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.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 69% biodegradation after 14 and 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.

Any other information on results incl. tables

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.10	2.10	2.10	2.45	2.10	2.10	2.10	2.10	2.22	2.10	2.33	2.10	2.10	2.10
2	4.87	-	7.66	-	16.01	-	10.09	-	6.85	-	5.22	-	21.92	-
6	9.34	-	13.61	-	34.14	-	10.84	-	21.45	-	15.68	-	44.06	-
8	9.40	-	19.15	-	36.92	-	33.37	-	46.78	-	28.09	-	56.87	-
10	14.48	-	19.95	-	43.09	-	41.15	-	49.93	-	40.81	-	62.47	-
14	20.29	-	20.74	-	42.95	-	39.55	-	44.43	-	46.47	-	62.33	-
21	26.93	-	26.93/24.00*	-	49.69	-	48.22	-	61.18	-	55.43	-	76.73	-
28	33.15/27.78**	-	35.84/27.55**	-	53.42	-	48.61	-	60.25	-	53.31	-	69.22	-
29	28.72	2.78	29.06	2.55	50.32	2.78	48.65	2.78	53.55	2.78	54.55	2.78	71.79	2.78

R = Replicate

Abs = CO2 absorber vessels

* = Result from analysis of frozen sample as original result deemed to be erroneous

** = Result from re-analysis of sample as original result deemed to be erroneous

Percentage BiodegradationValues

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	23	0	26
6	37	24	54
8	70	77	71
10	83	94	75
14	69	83	70
21	78	109	85
28	78	97	69
29*	69	84	72

* =  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:

readily biodegradable

Conclusions:

The test item attained 84% 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%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

Introduction

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 84% 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%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.