Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

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:

04 January 2021 to 29 April 2021

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)

Version / remarks:

July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Principles of method if other than guideline:

Study was performed according to a standard guideline.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 021015
Purity: 50.00% UB-20 ammonium salts; 50.00% Ethylene glycol
Physical State/Appearance: Clear brown liquid
Expiry Date: 24 November 2021
Storage Conditions: Approximately 4°C in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Inoculum:
A mixed population of activated sludge micro organisms was obtained on 22 March 2021 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK.


Preparation of Inoculum:
The activated sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excess Dissolved Organic Carbon (DOC). The washed sample was then continuously aerated at ~21ºC and used on the day of collection. Determination of the suspended solids level of the activated sludge was conducted by filtering a sample (100 mL) of the washed activated sludge by suction through a pre weighed Whatman GF/A filter paper using a Buchner funnel. Filtration 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 ~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 2.3 g/L prior to use.

Duration of test (contact time):

35 d

Initial conc.:

18.2 mg/L

Based on:

test mat.

Remarks:

Equivalent to 9.4 mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test item preparation:
Since the test item corresponded to 50.0% UB-20 ammonium salts in 50.0% ethylene glycol, two solvent control vessels were prepared containing the same concentration of ethylene glycol as the test item vessels. Based on preliminary solubility work, test item preparation into test systems was via the direct addition method. Since test item was supplied in solvent, use of ultrasonication, high shear mixing or solvents to aid was considered not appropriate, test item dispersion, due to risk of losing some of the ethylene glycol that the test item was supplied in. Test item (54.6 mg) was dispersed directly into the test vessel containing inoculated mineral medium, in duplicate, with the aid of mineral medium to wash the test item from the weighing receptacle. The volume was adjusted to 3 L to give a final concentration of 18.2 mg/L, equivalent to 9.4 mg carbon/L.


Reference item preparation:
A reference item stock solution (1000 mg/L) was prepared by dissolving sodium benzoate (1000 mg) directly in mineral medium and the volume adjusted to 1 L. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 L to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L.


Toxicity control:
Test item (54.6 mg) was dispersed directly into the test vessel containing inoculated mineral medium with the aid of mineral medium to wash the test item from the weighing receptacle. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 L, giving a final concentration of 18.2 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.


Test system preparation:
The following were prepared and inoculated in 5 L test culture vessels, each containing 3 L of preparation:
a) An inoculated control (in duplicate), consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate) (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 9.4 mg carbon/L.
d) A toxicity control (one vessel only), consisting of the test item plus the reference item in inoculated mineral medium to give a final concentration of 19.4 mg carbon/L.
e) A solvent control (in duplicate), containing ethylene glycol at the same concentration as the test item in inoculated mineral medium.

Ethylene glycol (27.3 mg, equivalent to the same concentration present in the test item vessel) was added to each solvent control vessel in to correct for any biodegradation from ethylene glycol in the test item vessels. Test vessels were inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids/L. The test was conducted in a temperature-controlled room at approximately 21 to 24°C for 35 days (temperature recorded daily from a vessel incubated alongside the test containing 3 L of reverse osmosis water), in darkness. Approximately 24 hours prior to addition of test and reference items, vessels were filled with 2400 mL of mineral medium and 39.1 mL of inoculum and aerated overnight. On Day 0, the test and reference items were added and the pH of all vessels measured (via Hach HQ30d Flexi handheld meter), and pH of the medium adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid solution if necessary, prior to the volume in all of the vessels being adjusted to 3 L by the addition of mineral medium, which had been purged overnight with CO2 free air. Inoculum control vessels were prepared similarly, without the addition of test item or reference item. Solvent control vessels were prepared as the inoculum control vessels with the addition of ethylene glycol. 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 produced by degradation was collected in two Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using deionized reverse osmosis water.

Test vessels were acidified on Day 35 to kill the micro organisms present and drive off any dissolved CO2 present in the test vessels and was followed by final biodegradation (CO2) analyses on Day 36.

Reference substance:

benzoic acid, sodium salt

Reference substance:

ethylene glycol

Preliminary study:

N/A

Test performance:

All validity criteria given in the OECD Test Guidelines were met. The total CO2 evolution in the inoculum control vessels on Day 28 was 34.48 mg/L, the inorganic carbon (IC) content of the test item suspension in the mineral medium at the start of the test (before dosing) was below 5% of the total carbon (TC) content, the difference between the values for CO2 production at the end of the test for the replicate vessels was <20%.


Total organic carbon of the diluted sodium benzoate stock solution confirmed that it had been prepared correctly.


The toxicity control attained 65% biodegradation after 14 days, 61% biodegradation after 28 days and 73% biodegradation after 35 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro organisms used in the test.


The TC and IC values in the culture vessels on Day 0, the pH values of the test preparations on Days 0 and 35, and observations made on the contents of the test vessels are presented under ‘Any other information on results incl. tables’.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

50

Sampling time:

28 d

Remarks on result:

other: Based on biodegradation of the ammonium salts of the UB-20 component of the test item

Parameter:

% degradation (CO2 evolution)

Value:

66

Sampling time:

36 d

Remarks on result:

other: Based on biodegradation of the ammonium salts of the UB-20 component of the test item

Details on results:

Results of IC analysis of samples from the first absorber vessels on Day 36 showed an increase in all replicate vessels with the exception of procedure control Replicate 1 and test item Replicate 2. The IC analysis of the samples from the second absorber vessels on Day 36 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.


Biodegradation of the ammonium salts of the UB-20 component of the test item attained 50% biodegradation after 28 days and 66% biodegradation after 35 days. Since degradation did not reach the 60% threshold within 28 days, the ammonium salts of UB-20 component of the test item does not qualify for classification as readily biodegradable. However, exceedance of 60% biodegradation under the conditions of a ready biodegradability test following extension of the incubation to 35 days signifies that the ammonium salts of UB 20 component is not persistent (P) or very persistent (vP) according to EU definitions. The ammonium salts of UB-20 cannot therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B, but can be considered to be inherently biodegradable according to the revised introduction to the OECD guidelines for the testing of Chemicals, Section 3.


Solvent control vessels showed substantial and rapid biodegradation during the first half of incubation, before reaching a plateau around Day 14. The degradation curve for the test item is distinctly biphasic, with an initial phase of CO2 production that ended at Day 14, followed by a second one that began just ahead of Day 28. The qualitative similarity of the events up to Day 14 strongly suggests that the IC production in the test item vessels during this phase of the study was dominated by biodegradation of the ethylene glycol solvent component. Quantitatively, however, cumulative IC production in the test item vessels progressively increased above that recorded in the solvent controls over this period. Since it is not possible to discriminate between IC contributions from different sources, the reason for the increase is unclear. However a plausible explanation for the above-solvent excess IC yield from the test item up to Day 14 is that this represents the partial shortening of the major hexyl and nonenyl carboxylate chains of the ammonium salts of UB-20 component, with the tertiary branched residue requiring a longer microbial adaptation that resulted in the second phase of biodegradation that began just before Day 28.

Results with reference substance:

Sodium benzoate attained 89% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window, thus confirming the suitability of the inoculum and test conditions and satisfying the validation criterion given in the OECD Test Guidelines. After 28 days 76% biodegradation was attained with 91% biodegradation after 35 days. The slight decrease in biodegradation between Days 14 and 28 was considered to be due to sampling/analytical variation.

Inorganic Carbon Values on Each Analysis Occasionfor the Inoculum Control and Procedure Control Vessels

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control
	R1		R2		R1		R2
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.40	1.52	1.52	1.52	1.40	1.75	1.63	1,63
2	6.61	-	1.52	-	1.40	-	1.63	-
6	14.30	-	7.31	-	21.92	-	23.43	-
8	15.59	-	13.38	-	34.37	-	34.25	-
10	18.81	-	16.17	-	37.50	-	35.20	-
14	21.31	-	18.24	-	39.44	-	38.88	-
21	25.46*/22.87	-	24.56*/20.62	-	49.80*/36.62**/48.78	-	46.19*/52.84**/48.00	-
28	29.46	-	26.99	-	51.07		50.74	-
31	26.83/32.62***	-	35.63/30.84***	-	46.98/54.55***	-	35.18/55.00***	-
35	34.20	-	30.65	-	60.87	-	57.33	-
36	34.43	1.85	31.02	2.09	60.39	2.44	59.18	1.74

R=     Replicate

Abs =   CO₂ absorber vessel

- =     No value determined

*        Result not used for calculation of the biodegradation value due to failure of the front calibration check on the run therefore sample re-analyzed.

**     Result of frozen sample not used for calculation of the biodegradation value due to inconsistent result compared to previous and/or subsequent results

***    Result from frozen sample as original result could not be used due to incorrect allocation of the calibration curve used to calculate the results

 

 

Inorganic Carbon Values on Each Analysis Occasion for the Solvent Control, Test Item and Toxicity Control Vessels

Day	Inorganic Carbon (mg IC)
	Solvent Control				Test ItemUB20 (50%) which is a 50:50 combination of ammonium salts of UB-20 and ethylene glycol				Toxicity Control
	R1		R2		R1		R2		R1
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.63	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.63
2	11.60	-	11.02	-	12.53	-	12.64	-	28.54	-
6	22.26	-	21.22	-	28.60	-	28.83	-	49.13	-
8	23.51	-	22.13	-	31.65	-	32.45	-	56.07	-
10	25.99	-	25.20	-	34.54	-	37.85	-	62.13	-
14	29.92	-	26.63	-	41.59	-	42.05	-	65.85	-
21	30.19*/30.31	-	29.74*/28.50	-	40.79*/41.91	-	40.79^*/47.55**/43.71	-	64.56^*/53.97^**/71.66	-
28	33.04	-	34.27	-	47.49	-	48.05	-	69.33	-
31	23.71/33.73***	-	38.63/35.74^***	-	44.76/49.21***	-	46.65/51.33^***	-	73.48/71.81^***	-
35	37.96	-	40.28	-	54.89	-	59.65	-	80.79	-
36	39.49	2.44	40.59	2.44	57.31	3.13	59.51	2.44	81.62	3.13

R =   Replicate

Abs =   CO₂ absorber vessel

- =    No value determined

*       Result not used for calculation of the biodegradation value due to failure of the front calibration check on the run therefore sample re-analyzed.

**     Result of frozen sample not used for calculation of the biodegradation value due to inconsistent result compared to previous and/or subsequent results

***   Result from frozen sample as original result could not be used due to incorrect allocation of the calibration curve used to calculate the results

 

 

Average inorganic carbon values on each analysis occasion for the inoculum control, solvent control, and test item vessels

Day	Inorganic Carbon (mg IC)
	Inoculum Control		Procedure Control		Solvent Control		Test Item UB20-LDB (50%) which is a 50:50 combination of ammonium salts of UB-20 and Ethylene Glycol
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.46	1.52	1.52	1.69	1.69	1.75	1.75	1.75
2	4.07	-	1.52	-	11.31	-	12.59	-
6	10.81	-	22.68	-	21.74	-	28.72	-
8	14.49	-	34.31	-	22.82	-	32.05	-
10	17.49	-	36.35	-	25.60	-	36.20	-
14	19.78	-	39.16	-	28.28	-	41.82	-
21	21.75	-	48.39	-	29.41	-	42.81	-
28	28.23	-	50.91	-	33.66	-	47.77	-
31	31.73	-	54.78	-	34.74	-	50.27	-
35	32.43	-	59.10	-	39.12	-	57.27	-
36	32.73	1.97	59.79	2.09	40.04	2.44	58.41	2.79

Abs = CO₂ absorber vessel

- = No value determined

 

 

Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Item*	Toxicity Control
0	0	0	0
2	52	5	30
6	68	25	47
8	68	33	57
10	69	38	63
14	89	48	65
21	89	48	73
28	76	50	61
31	77	55	64
35	89	65	72
36**	91	66	73

* Biodegradation values for the ammonium salts of UB-20 component in the test item calculated after correcting for the inorganic carbon values of the solvent control vessels which contained ethylene glycol at the same concentration as was nominally present in the test item.

**Day 36 values corrected to include any carry-over of CO₂ detected in Absorber 2

 

 

Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/L)**	Inorganic Carbon*(mg/L)	IC Content (% of TC)
Test Item*** 9.4 mg C/L R1	9.61	0.24	3
Test Item*** 9.4 mg C/L R2	9.59	0.22	2

R = Replicate

*   = Corrected forsolventcontrol values. Negative values are due to measured concentrations being less thansolventcontrol values.

** = TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of thetest item

*** = UB20 -LDB (50%) which is a 50:50 combination of ammonium salts of UB-20 and ethylene glycol

 

 

pH Values of the Test Preparations on Days 0 and 35

Test Vessel	pH
	Day 0 Pre-Adjustment	Day 0 Post-Adjustment	Day 35
Inoculum ControlR1	7.7	7.5	7.5
Inoculum Control R2	7.7	7.5	7.4
Procedure Control R1	7.7	7.5	7.5
Procedure Control R2	7.7	7.5	7.5
Solvent Control R1	7.7	7.5	7.4
Solvent Control R2	7.7	7.5	7.4
Test Item R1	7.6	-	7.3
Test Item R2	7.6	-	7.4
Toxicity Control	7.6	-	7.3

R = Replicate

-  = No adjustment necessary

 

 

Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 7	Day 14	Day 21	Day 28	Day 35
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Solvent Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Test Item	R1	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	R2	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
Toxicity Control		Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible

R = Replicate 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The biodegradation of the ammonium salts of UB-20 attained 50% and 66% biodegradation after 28 and 35 days, respectively. Since degradation did not reach the 60% threshold within 28 days, the ammonium salts of UB-20 do not qualify for classification as readily biodegradable. However, exceedance of 60% biodegradation under the conditions of a ready biodegradability test following extension of the incubation to 35 days signifies that the ammonium salts of UB-20 are not persistent (P) or very persistent (vP) according to EU definitions. The ammonium salts of UB-20 are considered to be inherently biodegradable according to the revised introduction to the OECD guidelines for the testing of Chemicals, Section 3.

Executive summary:

The ready biodegradability of UB20-LDB (50%) was assessed according to OECD Guideline 301B (Ready Biodegradability: CO2 Evolution Test), EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test) and EPA OPPTS 835.3110 (Ready Biodegradability).

 

The test item, at a concentration of 9.4 mg carbon/L, was exposed to activated sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 21 to 24°C for 35 days. The study was extended from 28 days to 35 days to determine if greater than 60% biodegradation could occur. Due to technical necessity, ammonium salts of UB‑20 was supplied and presented to the test system as the test item UB20-LDB (50%) - a 50:50 solution of ammonium salts of UB-20 in ethylene glycol. For this reason, the test design included an additional solvent control containing ethylene glycol alone. The performance of the ammonium salts of UB‑20 was determined by correcting the CO₂ recovery from UB20-LDB (50%) by subtraction of the contemporary CO₂ yield in the solvent control. Biodegradation was assessed by the determination of CO2 produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

All validity criteria given in the OECD Test Guidelines were met and the study is considered reliable. The toxicity control attained 65% biodegradation after 14 days, 61% biodegradation after 28 days and 73% biodegradation after 35 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The reference item (sodium benzoate) attained 89% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window, thus confirming the suitability of the inoculum and test conditions.

 

The biodegradation of the ammonium salts of UB‑20 determined by difference (as outlined above) attained 50% after 28 days and 66% biodegradation after 35 days. Since degradation did not reach the 60% threshold within 28 days, the ammonium salts of UB‑20 do not qualify for classification as readily biodegradable. However, exceedance of 60% biodegradation under the conditions of a ready biodegradability test following extension of the incubation to 35 days signifies that the ammonium salts of UB‑20 are not persistent (P) or very persistent (vP) according to EU definitions. The ammonium salts of UB-20 are considered to be inherently biodegradable according to the revised introduction to the OECD guidelines for the testing of Chemicals, Section 3.

Description of key information

Ready biodegradability

The ready biodegradability of UB20-LDB (50%) was assessed according to OECD Guideline 301B (Ready Biodegradability: CO2 Evolution Test), EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test) and EPA OPPTS 835.3110 (Ready Biodegradability).

 

The test item, at a concentration of 9.4 mg carbon/L, was exposed to activated sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 21 to 24°C for 35 days. The study was extended from 28 days to 35 days to determine if greater than 60% biodegradation could occur. Due to technical necessity, ammonium salts of UB‑20 was supplied and presented to the test system as the test item UB20-LDB (50%) - a 50:50 solution of ammonium salts of UB-20 in ethylene glycol. For this reason, the test design included an additional solvent control containing ethylene glycol alone. The performance of the ammonium salts of UB‑20 was determined by correcting the CO₂ recovery from UB20-LDB (50%) by subtraction of the contemporary CO₂ yield in the solvent control. Biodegradation was assessed by the determination of CO2 produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

All validity criteria given in the OECD Test Guidelines were met and the study is considered reliable. The toxicity control attained 65% biodegradation after 14 days, 61% biodegradation after 28 days and 73% biodegradation after 35 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The reference item (sodium benzoate) attained 89% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window, thus confirming the suitability of the inoculum and test conditions.

 

The biodegradation of the ammonium salts of UB‑20 determined by difference (as outlined above) attained 50% after 28 days and 66% biodegradation after 35 days. Since degradation did not reach the 60% threshold within 28 days, the ammonium salts of UB‑20 do not qualify for classification as readily biodegradable. However, exceedance of 60% biodegradation under the conditions of a ready biodegradability test following extension of the incubation to 35 days signifies that the ammonium salts of UB‑20 are not persistent (P) or very persistent (vP) according to EU definitions. The ammonium salts of UB-20 are considered to be inherently biodegradable according to the revised introduction to the OECD guidelines for the testing of Chemicals, Section 3.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater