Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2 yl)phenoxy]methyl}oxirane and 2RS)-2-({4-[2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16th April 2020 to 29th June 2020

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical appearance : Solid
Purity as per Certificate
of Analysis
: 60 -85% (No purity correction during dose
formulation preparation)
Total Organic Content
(TOC)
: 62.3 %
Batch No. : GRM193K01
Manufactured date : 19 July 2019
Expiry date : 03 September 2021
Physico-chemical
properties
: Density: 1.32 g/cm3 (23 °C)
Storage conditions : Ambient (+15 to +25ºC)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Inoculum
The inoculum was secondary effluent, collected from a treatment plant
receiving predominantly domestic sewage. This effluent was used as test
system as it is recommended in the guideline. A fresh sample of secondary
effluent was collected from the treatment plant and was kept aerobic during
transport.
This effluent was allowed to settle for one hour, decanted and the decanted
effluent was used in the test.

Source of the Inoculum
Sewage Treatment Plant
Eurofins Advinus Limited
Bengaluru – 560 058
India
Determination of Bacterial Population in the Inoculum
The bacterial population in the inoculum was determined as colony forming
units (CFU/mL) by diluting the inoculum to an appropriate dilution and then
plating on nutrient agar plates.
Preconditioning of the Inoculum
The decanted effluent was preconditioned by aerating for 6 days at 22.4 to
23.8°C.

Duration of test (contact time):

29 d

Initial conc.:

25 mg/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Outline of the Method
A measured volume of the inoculated mineral medium, containing a known
concentration of the test item [10-20 mg total organic carbon (TOC) per litre]
as the nominal sole source of organic carbon was aerated by the passage of
carbon dioxide-free air at a controlled rate in the dark. Degradation was
followed over 28 days by determining the carbon dioxide produced. The
carbon dioxide was trapped in barium hydroxide and was measured by
titration of the residual hydroxide or as inorganic carbon. The amount of
carbon dioxide produced from the test item (corrected for that derived from
the blank inoculum) was expressed as a percentage of theoretical carbon
dioxide (ThCO2).
Test Item Solubility
The test item was tested for its solubility in mineral media at 250 mg/L.
Preparation of Test Medium
The stock solutions and the test medium were prepared as per the
compositions of chemicals given in Appendix 1. High quality ultra-pure
water delivered by a Milli-Q system was used to prepare the stock solutions
and the mineral medium.

To each 5 L flask, 2400 mL of mineral medium was added and mixed with
300 mL of the pre-conditioned inoculum. A separate 3000 mL of mineral
medium was also prepared in a flask to use it for further dilutions.
A sample of the mineral medium was checked for the inorganic carbon
content.
These flasks were aerated with CO2 free air at 100 mL/minute, overnight to
purge the system of carbon dioxide.
Inoculum Blanks
To the test flasks numbered 3 and 4 (inoculum blanks), 300 mL each of
mineral medium (previously aerated with CO2-free air) was added to make
the total suspension volume to 3000 mL in each flask.

Addition of Test Item and Reference Item
The total organic carbon (TOC) in the test as well as the reference item was
determined using the formula:
% TOC = Carbon content of the test/reference item Molecular weight of the test/reference item x 100
Molecular formula of reference item (Sodium benzoate) – C7H5NaO2
Based on the TOC, 75 mg of test item was weighed and made up to 300 mL
using mineral medium (previously aerated with CO2-free air) and added to
each test flasks 1 and 2 separately. Similarly, 78 mg of reference item was
made up to 300 mL with mineral medium (previously aerated with CO2-free
air) and added to test flask 5. A quantity of 38 mg test item and 39 mg of the
reference item was mixed and made up to 300 mL using mineral medium
(previously aerated with CO2-free air) and added to test flask 6. Final volume
in the test flask was 3000 mL.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

47.64

Sampling time:

28 d

Details on results:

The mean carbon dioxide produced from the inoculum blank (Flask No.
3 and 4) on days 2, 5, 7, 9, 12, 15, 19, 23, 27 and 29 after the treatment was
8.80, 10.18, 8.64, 35.04, 7.65, 9.90, 11.11, 11.33, 5.61 and 7.32 mg,
respectively. The total carbon dioxide produced from the inoculum blank for
throughout the test period was 38.53 mg/L.
The cumulative carbon dioxide produced from the test item was 7.48, 20.84,
37.94, 31.83, 51.35, 55.53, 65.76, 73.68, 80.94 and 84.41 mg in Flask 1 on
days 2, 5, 7, 9, 12, 15, 19, 23, 27 and 29 after the treatment, respectively.
Similarly, in Flask 2 it was 6.05, 20.29, 34.31, 28.31, 45.30, 50.47, 58.50,
67.08, 77.31 and 79.24 mg on days 2, 5, 7, 9, 12, 15, 19, 23, 27 and 29 after
the treatment, respectively.
The cumulative carbon dioxide produced from the reference item (Flask No.
5) was 23.65, 59.01, 85.02, 75.72, 118.12, 143.20, 160.03, 164.21, 169.38
and 162.07 mg, on days 2, 5, 7, 9, 12, 15, 19, 23, 27 and 29 after the
treatment, respectively.
The cumulative carbon dioxide produced from the toxicity control (Flask
No. 6) was 16.61, 29.97, 42.78, 42.06, 57.84, 70.38, 80.61, 87.87, 93.92 and
86.61 mg, on days 2, 5, 7, 9, 12, 15, 19, 23, 27 and 29 after the treatment,
respectively.

The pH of the test solutions at the end of the test was 8.08, 8.10, 8.34, 8.19,
8.56 and 8.49 in Flask 1, 2, 3, 4, 5 and 6 respectively.

TABLE 1.      Carbon-dioxide Production

Day	CO2Produced (mg)							Cumulative CO2produced (mg) Test – Blank Mean
	Test Flasks				Blank
	Flask 1	Flask 2	Flask 5	Flask 6	Flask 3	Flask 4	Mean	Flask 1	Flask 2	Flask 5	Flask 6
2	16.28	14.85	32.45	25.41	8.25	9.35	8.80	7.48	6.05	23.65	16.61
5	23.54	24.42	45.54	23.54	10.67	9.68	10.18	20.84	20.29	59.01	29.97
7	25.74	22.66	34.65	21.45	8.25	9.02	8.64	37.94	34.31	85.02	42.78
9	28.93	29.04	25.74	34.32	35.97	34.10	35.04	31.83	28.31	75.72	42.06
12	27.17	24.64	50.05	23.43	7.04	8.25	7.65	51.35	45.30	118.12	57.84
15	14.08	15.07	34.98	22.44	11.55	8.25	9.90	55.53	50.47	143.20	70.38
19	21.34	19.14	27.94	21.34	11.44	10.78	11.11	65.76	58.50	160.03	80.61
23	19.25	19.91	15.51	18.59	11.88	10.78	11.33	73.68	67.08	164.21	87.87
27	12.87	15.84	10.78	11.66	5.94	5.28	5.61	80.94	77.31	169.38	93.92
29	10.78	9.24	0.00	0.00	8.25	6.38	7.32	84.41	79.24	162.07	86.61

 

Total CO₂produced in the inoculum blank during the test   =115.575 mg/3L                                                                                                                                                                           

                                                                                               =38.53 mg/L

Note: 

Flask 1 and 2: Contain test item (Bisphenol C Epoxy) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (Sodium benzoate) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

TABLE 2.      pHof Test Solutions

Flask No.	pH at the end of the test (Day 28)
Flask 1	8.08
Flask 2	8.10
Flask 3	8.34
Flask 4	8.19
Flask 5	8.56
Flask 6	8.49

 

 

Note: 

Flask 1 and 2: Contain test item (Bisphenol C Epoxy) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (Sodium benzoate) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

TABLE 3.      Degradation of Test Item and Reference Item

Day	Test Item			Reference Item	Test Item + Reference Item
	Flask 1	Flask 2	Mean
2	4.36	3.52	3.94	14.13	9.73
5	12.13	11.81	11.97	35.26	17.56
7	22.09	19.98	21.04	50.80	25.07
9	18.53	16.48	17.51	45.25	24.65
12	29.90	26.37	28.14	70.58	33.89
15	32.33	29.38	30.86	85.57	41.24
19	38.29	34.06	36.18	95.62	47.24
23	42.90	39.06	40.98	98.12	51.49
27	47.12	45.01	46.07	101.21	55.04
29	49.14	46.13	47.64	96.84	50.75

 

% degradation	=	mg CO2produced	x	100
		mg TOC added in test x 3.67

 

 

Note: 

Flask 1 and 2: Contain test item (Bisphenol C Epoxy) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (Sodium benzoate) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The mean per cent degradation of test item was 47.64% at the end of test while, the percent degradation of reference item was 96.84% and the toxicity control was 50.75% at the end of the test.

The substance is not considered rapidly biodegradable, as only 47.64% mean degradation was achieved within the 28-day period of the test.

Executive summary:

The ready biodegradability of Bisphenol C Epoxy was tested using the CO₂Evolution Test. The test item was added to two test vessels at the concentration of 25 mg/L (equivalent to 15.6 mg of Total Organic Carbon/L). Two flasks as controls containing only the inoculum, one flask as procedure control containing reference item and one flask as toxicity control containing the test item and the reference item were also included in test. All the treatments were added with equal volume of inoculum which was collected from the secondary effluent treatment plant receiving predominantly domestic sewage and volume made with mineral media.

Treatment mixtures were aerated for 29 days with carbon dioxide (CO₂) free air. The CO₂released was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on days 2, 5, 7, 9, 12, 15, 19, 23, 27 and 29 after the initiation of the test.

The mean per cent degradation of test item was 47.64% at the end of test while, the percent degradation of reference item was 96.84% and the toxicity control was 50.75% at the end of the test.

The test fulfilled all the validity criteria.

 

Based on the results, the test item was concluded asinherentlybiodegradable since only 47.64% mean degradation was achieved within the 28-day period of the test.

Description of key information

The substance is not considered rapidly biodegradable, as only 47.64% mean degradation was achieved within the 28-day period of the test.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater