Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 November 2014 and 24 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test System
A mixed population of activated sewage sludge micro-organisms was obtained on
24 November 2014 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 sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of DOC that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

other: carbon

Details on study design:

Preliminary Investigational Work
During the test, samples are taken for DOC analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analyzed for DOC using a Shimadzu TOC-VCPH TOC analyzer (see Appendix 3).

A nominal amount of test item (100 mg) was dissolved in mineral medium (1000 mL) to give a
100 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further nominal amount of test item (100 mg) was dissolved in mineral medium and inoculated at a concentration of 300 mg suspended solids (ss)/L prior to adjusting to a final volume of 1000 mL. Two samples were taken for DOC analysis; one after filtration through a Gelman 0.45 µm AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 300 mg ss/L and then filtering or centrifuging as per the test item samples.

A mixed population of activated sewage sludge micro-organisms was obtained on
17 November 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Experimental Preparation
Test Item
The test item was dissolved directly in reverse osmosis purified deionized water prior to dispersal in inoculated mineral medium to give the required test concentration.

A nominal amount of test item (1500 mg) was dissolved in reverse osmosis purified deionized water with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) and the volume adjusted to 3 liters to give a 500 mg/L stock solution. An aliquot (672 mL) of the 500 mg/L stock solution was dispersed in inoculated mineral medium and the volume adjusted to 2 liters to give a final test concentration of 168 mg/L, equivalent to 100 mg carbon/L.

The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the solution.
A test concentration of 100 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item
A reference item, diethylene glycol, was used to prepare the procedure control vessels. An initial stock solution of 2000 mg/L was prepared by dissolving the reference item directly in reverse osmosis purified deionized water with the aid of ultrasonication for approximately
5 minutes. An aliquot (220 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 2 liters to give a final concentration of 220 mg/L, equivalent to 100 mg carbon/L. The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.


Toxicity Control
A toxicity control, containing the test item and diethylene glycol was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An aliquot (672 mL) of the 500 mg/L test item stock solution was dispersed in inoculated mineral medium along with an aliquot (220 mL) of the 2000 mg/L reference item stock solution. The volume was adjusted to 2 liters to give a final concentration of 168 mg test item/L, plus
220 mg diethylene glycol/L, equivalent to a final concentration of 200 mg carbon/L.


Preparation of Test System
The following test solutions were prepared and inoculated in 3 liter glass test vessels each containing 2 liters of solution:

a) An inoculum control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (diethylene glycol), in duplicate, in inoculated mineral medium to give a final concentration of 100 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 100 mg carbon/L.
d) The test item plus diethylene glycol, one replicate, in inoculated mineral medium to give a final concentration of 200 mg carbon/L to act as a toxicity control.

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 300 mg suspended solids/liter in order to give an inoculum to carbon ratio of 3:1.
The test vessels were covered with polypropylene lids to reduce evaporation and incubated at temperatures of between 20 and 25 °C in darkness for 28 days. The test vessels were constantly aerated with compressed air via narrow bore glass tubes and stirred constantly by a magnetic.

Evaluations
DOC Analysis
Samples (approximately 30 mL) were filtered through 0.45 µm Gelman AcroCap disposable filters. The first approximate 5 mL of filtrate was discarded.

Samples were taken for analysis at 0 and 3 hours and on Days 1, 8, 14, 21, 27 and 28.

Prior to sampling for analysis any losses due to evaporation were corrected by the addition of deionized water and any material adhering to the culture vessel resuspended.

The samples were analyzed for DOC using a Shimadzu TOC-VCPH TOC analyzer. Samples
(50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyzer. Total Carbon analysis is carried out at 680 ºC using a platinum catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involved conversion by 25% orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.


Water Quality Criteria
Immediately prior to sampling the temperature and dissolved oxygen concentration (by means of a Hanna Instruments HI 93510 digital thermometer and a Hach HQ40d Flexi handheld meter of each vessel were recorded. The pH of each vessel was also determined using a Hach HQ40d Flexi handheld meter and adjusted to pH 7 to 8, where necessary, with sodium hydroxide or hydrochloric acid solutions.


Data Evaluation
Calculation of Carbon Content
The test item contains 59.58% carbon (data supplied by the Sponsor) and so for a concentration of 100 mg C/L the total organic carbon present was 200 mg C.

The theoretical amount of carbon present in the reference item, diethylene glycol (C4H10O3) was calculated as follows:

((No of C atoms x mol wt of C) / mol wt of diethylene glycol) x 100
= ((4 x 12.011) / 106.12) x 100
=45.27 %

Thus for a test concentration of 220 mg/L the theoretical carbon concentration was approximately 100 mg C/L.
Percentage Biodegradation or Loss of DOC
The percentage biodegradation or loss of DOC for each sample was calculated using the following equation:

Dt (%) = [1 – (Ct – Cbt) / (Co – Cbo)] x 100


Where:

Dt = Biodegradation or loss of DOC (%) at time t
Ct = mean mg DOC/L of sample at time t
Cbt = mean mg DOC/L of control (blank) at time t
Co = mean mg DOC/L of sample after 3 hours ± 30 minutes
Cbo = mean mg DOC/L of control (blank) after 3 hours ± 30 minutes

Validation Criteria
The results of the degradation test are considered valid if in the same test the reference item attains >70% degradation within 14 days.

Reference substance:

diethylene glycol

Parameter:

% degradation (DOC removal)

Value:

25

Sampling time:

28 d

Details on results:

Preliminary Investigational Work
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.


Definitive Test
From DOC analysis the test item attained 25% biodegradation after 28 days.

OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Summary of Considerations in the Report from the OECD Expert Group on Degradation and Accumulation” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralisation may be regarded as evidence for ultimate biodegradation.

Results with reference substance:

The procedure control, Diethylene glycol attained 100% biodegradation after 14 days and 28 days, thereby confirming the suitability of the inoculum and culture conditions.

The toxicity control attained 54% biodegradation after 14 days and 64% biodegradation after 28 days, therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.

The pH values did not fall below 6.9 in any test vessel during the study and were readjusted to pH 7 to 8 where necessary on sampling days. Aerobic conditions in the test vessels were maintained throughout the study as dissolved oxygen concentrations remained at or above
5.8 mg O2/L in all culture vessels.

Dissolved Organic Carbon (DOC) Values on Each Sampling Occasion

Vessel		DOC (mg C/L)
		Day
		0 Hours	3 Hours	1	8	14	21	27	28
Inoculum Control	R1	1.84	1.80	2.03	3.02	2.87	3.44	3.72	3.67
	R2	1.43	1.33	1.90	3.32	2.90	3.18	3.50	3.68
	Mean	1.64	1.57	1.97	3.17	2.89	3.31	3.61	3.68
Procedure Control	R1	102.40	103.30	105.10	3.05	3.09	2.79	3.11	3.22
	R2	105.70	107.10	108.10	3.27	3.27	3.07	3.08	3.12
	Mean	104.05	105.20	106.60	3.16	3.18	2.93	3.10	3.17
Test Item	R1	94.55	95.16	96.93	97.46	93.12	89.33	76.06	74.85
	R2	94.99	95.25	96.88	97.79	93.70	85.69	73.32	72.35
	Mean	94.77	95.21	96.91	97.63	93.41	87.51	74.69	73.60
Toxicity Control		202.60	203.30	207.90	97.76	96.61	92.08	77.72	76.34

R₁- R₂= Replicates 1 and 2

Percentage Biodegradation / DOC Loss Values

Day	% Biodegradation / DOC Loss
	Procedure Control	Test Item	Toxicity Control
1	0	0	0
8	100	0	53
14	100	3	54
21	100	10	56
27	100	24	63
28	100	25	64

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test item attained 25% biodegradation after 28 days.

OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Summary of Considerations in the Report from the OECD Expert Group on Degradation and Accumulation” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralization may be regarded as evidence for ultimate biodegradation.

The test item can therefore be considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Executive summary:

Introduction

The study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 302B, “Inherent Biodegradability; Modified Zahn-Wellens/EMPA Test”, Method C.9 of Commission Regulation (EC) No. 440/2008, and the US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3200.

 

 

Methods…….

The test item was exposed to activated sewage sludge micro-organisms at a concentration of 100 mg carbon/L with mineral medium in the dark at temperatures of between 20 and 25 ºC for 28 days. The biodegradation of the test item was assessed by the determination of dissolved organic carbon removal. Control cultures with inoculum and the reference item, diethylene glycol, together with a toxicity control were used for validation purposes.

 

 

Results…….

The test item attained 25% biodegradation after 28 days.

 

OECD Guideline No 302B does not give any definitive pass levels for test items, however the “Summary of Considerations in the Report from the OECD Expert Group on Degradation and Accumulation” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralisation may be regarded as evidence for ultimate biodegradation.

 

The test item can therefore be considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 29th September 2014 and 27th October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test System
A mixed population of sewage treatment micro-organisms was obtained on 23 September 2014 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

None

Reference substance:

aniline

Preliminary study:

None

Test performance:

Validation Criteria:
The mean BOD of the inoculated mineral medium (control) was 31.26 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The pH of the inoculated test item vessels on Day 28 ranged from 7.8 to 7.9 and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Key result

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

28 d

Remarks on result:

other: No St. dev.

Details on results:

Results:
Daily BOD values for the test item, procedure control, toxicity control and inoculum control vessels are given in Table 1*. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2*. Biodegradation curves are given in figure 3* respectively. The pH values of each individual vessel on Days 0 and 28 are given in Table 3*.

*Tables 1 -3 can be found in the any other information on resilts section, figure 3 can be found in theillustration section of Iuclid.

Biodegradation:
The test item attained 8% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

The toxicity control attained 39% biodegradation after 14 days and 43% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Aniline (procedure control) attained 74% biodegradation after 14 days and 79% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Results with reference substance:

Aniline (procedure control) attained 74% biodegradation after 14 days and 79% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1     Biological Oxygen Demand (BOD) Values:

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test Item		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	0.00	0.00	0.00	0.00	0.00	0.38
2	0.16	0.00	0.00	1.88	1.42	2.66
3	2.70	0.00	2.24	10.04	9.38	6.04
4	4.28	0.00	7.04	11.66	10.92	12.50
5	5.50	0.50	93.04	13.04	12.28	100.70
6	7.24	2.30	180.52	15.82	14.92	164.44
7	8.12	3.54	199.54	17.40	16.16	171.88
8	8.92	4.66	213.50	18.74	17.16	177.10
9	10.04	6.16	222.92	20.28	18.54	181.80
10	10.74	7.70	229.12	21.36	19.62	187.54
11	12.04	9.28	235.08	22.62	21.00	196.00
12	13.08	10.74	239.40	23.86	22.32	203.08
13	14.42	12.46	242.36	25.54	24.00	208.66
14	15.46	13.86	244.52	26.90	25.08	212.66
15	16.62	15.20	246.40	28.36	26.20	216.08
16	17.86	16.36	248.66	29.90	27.54	218.92
17	18.86	17.58	251.02	31.40	28.48	221.28
18	19.86	18.82	253.60	32.94	29.66	223.50
19	20.70	19.82	255.98	34.32	30.58	225.50
20	21.74	21.08	258.32	35.98	32.16	227.70
21	23.24	22.66	260.98	38.10	34.02	230.44
22	24.40	24.08	263.18	39.82	35.66	232.78
23	25.58	25.54	265.48	41.60	37.28	235.32
24	26.44	26.62	267.36	43.10	38.52	237.40
25	27.20	27.78	268.98	44.32	39.40	239.32
26	28.48	29.32	271.14	46.36	40.90	242.02
27	29.90	30.62	273.18	48.32	42.64	244.70
28	30.86	31.66	274.52	49.52	43.70	246.60

 

R₁– R₂= Replicates 1 and 2

Table 2     Percentage Biodegradation Values:

Day	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	0	1	1	1	1
3	0	4	4	4	1
4	2	5	4	5	2
5	29	5	5	5	19
6	57	6	5	6	32
7	63	6	5	6	33
8	67	6	5	6	34
9	70	6	5	6	34
10	71	6	5	6	35
11	73	6	5	6	37
12	74	6	5	6	38
13	74	6	5	6	39
14	74	6	5	6	39
15	75	6	5	6	40
16	75	7	5	6	40
17	75	7	5	6	40
18	76	7	5	6	40
19	76	7	5	6	41
20	77	7	5	6	41
21	77	8	6	7	41
22	77	8	6	7	41
23	78	8	6	7	42
24	78	8	6	7	42
25	78	9	6	8	42
26	78	9	6	8	42
27	79	9	6	8	42
28	79	9	6	8	43

 

R₁– R₂= Replicates 1 and 2

Table 3     pH Values of the Test Preparationson Days 0 and 28:

 

Test Vessel	pH
	Day 0 Pre-Adjustment	Day 0 Post-Adjustment	Day 28
Inoculum ControlR1	7.8	7.4	8.1
Inoculum Control R2	7.8	7.4	7.8
Procedure Control	7.8	7.6	7.7
Test Item R1	7.6	-	7.8
Test Item R2	7.6	-	7.9
Toxicity Control	7.6	-	7.9

 

 R₁– R₂    =      Replicates 1 and 2

-                          =     No adjustment necessary

Theoretical Oxygen Demand Values:

Calculation of Theoretical Oxygen Demand (ThOD) for the test and reference items:

 

Test Item:                   1,3-Propanediol,2-ethyl-2-(hydroxymethyl)-, oligomeric reaction product with (chloromethyl)oxirane  C₁₅H₂₆O₆           mol wt = 302.37

 

ThOD (NO3) = 16[30 +13 - 6] / 302.37 = 1.96 mg O₂/mg.

 

Therefore for a test concentration of 100 mg/L, the ThOD will be 196 mg O₂/L. 

Reference Item (Procedure Control) :          Aniline C₆H₅NH₂       mol wt = 93.13

 

ThOD (NO3) = 16[12 + 3.5 + 2.5] / 93.13 = 3.09 mg O₂/mg

 

Therefore, for a test concentration of 100 mg/L, the ThOD will be 309 mg O₂/L.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 8% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Care should be taken in the interpretation of these results due to the possible inhibitory nature of the test item to the sewage treatment micro-organisms used in the study.

Executive summary:

Introduction:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

 

Methods:

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at approximately 23 ºC for 28 days. 

 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

 

Results:

The test item attained 8% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

 

 

Description of key information

Both readily biodegradation screening studies showed <10% degradation over the 28 day period. The test item cannot be considered readily biodegradable according to the OECD 301B or 301F studies.
The test item attained 25% biodegradation after 28 days according to OECD Guideline No 302B and therefore can be considered to be inherently, primarily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater