Reaction product of "Fatty acids, C16-18 (even numbered) and C18-unsaturated" and "reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 25 - October 26, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

A temporary breakdown in the aeration (< 1 day) was noted on nominal day 11. Evaluation: This breakdown was considered to have no effect on the outcome of these studies.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

A temporary breakdown in the aeration (< 1 day) was noted on nominal day 11. Evaluation: This breakdown was considered to have no effect on the outcome of these studies.

Principles of method if other than guideline:

ISO Standard 9439 "Water Quality - Evaluation of ultimate
aerobic biodegradability of organic compounds in aqueous
medium - carbon dioxide evolution test (1999).

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (38 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
- Pretreatment: Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Concentration of sludge: 1% final volume
- Initial cell/biomass concentration: The concentration of suspended solids was 4.1 g/l in the concentrated sludge
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

17 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock solutions of A)8.50 g KH2PO4
mineral components 21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

Mineral medium 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

- Test temperature: Continuously measured in a vessel with Milli-RO water in the same room.
- pH: Measured at the start of the test and on the 28th day.
- pH adjusted: no
- Aeration of dilution water: During the test period the test media were aerated and stirred continuously.
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration: Test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Positive control: containing reference substance and inoculum (1 bottle). Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Preparation: The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
-Method used to create aerobic conditions: The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Measuring equipment: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul), Merck, Darmstadt, Germany).
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency/method: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck, Darmstadt, Germany) was used as pH-indicator.
On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCl (37%, Merck, Darmstadt, Germany) was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
- Sample storage before analysis: The bottles were aerated overnight to drive off CO2 present in the test suspension.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum
- Toxicity control: containing test substance, reference substance and inoculum

Reference substance:

other: Sodium acetate

Test performance:

Theoretical CO2 production:
The TOC content of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was determined to be 69.98%. Based on the TOC content the ThCO2 of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was calculated to be 2.57 mg CO2/mg.
The ThCO2 per test bottle was 86.9 mg CO2 (bottle A), 87.6 mg CO2 (bottle B), 85.7 mg CO2 for the positive control and 173.3 mg CO2 for the toxicity control.

Biodegradation
All data are in the field 'Any other information on results incl. tables'. The results of CO2 production and biodegradation in each test bottle are listed in Tables 1-5.

The relative degradation values calculated from the measurements performed during the test period revealed 56 and 54% degradation of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation within a 10-day window) was not met. Based on the curves for degradation of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol, 60 % biodegradation would be reached after 30-31 days.

In the toxicity control more than 25% degradation occurred within 14 days (42%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 55

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance):
0 % degradation after 1 d
9 % degradation after 4 d
18 % degradation after 6 d
27 % degradation after 8 d
37 % degradation after 11 d
42 % degradation after 13 d
45 % degradation after 15 d
48 % degradation after 20 d
52 % degradation after 25 d
55 % degradation after 29 d

Results with reference substance:

Points of degradation plot (reference substance):
0 % degradation after 1 d
25 % degradation after 4 d
38 % degradation after 6 d
47 % degradation after 8 d
57 % degradation after 11 d
60 % degradation after 13 d
66 % degradation after 15 d
71 % degradation after 20 d
74 % degradation after 25 d
77 % degradation after 29 d

Table 1     CO₂production in the blank.   Day HCl (0.05 N) titrated (ml) Produced CO₂ (ml HCl) Produced CO₂ (mg) Cumulative CO₂ (mg)   Ba(OH)₂¹⁾ Blank (mean)   1 51.51 49.72 1.79 2.0 2.0   4 51.82 47.09 4.72 5.2 7.2   6 52.36 49.53 2.83 3.1 10.3   8 52.55 49.24 3.31 3.6 13.9   11 51.03 48.47 2.57 2.8 16.7   13 50.85 48.86 1.99 2.2 18.9   15 52.33 48.30 4.03 4.4 23.4   20 52.37 46.92 5.44 6.0 29.3   25 50.23 43.86 6.37 7.0 36.3   29 52.32 43.17 9.15 10.1 46.4   29 48.11 43.91 4.20 4.6 51.0   29 49.55 47.32 2.23 2.4 53.5   ¹⁾: "Strength" of untreated 0.0125 M Ba(OH)₂solution

	Table 2     CO2production and percentage biodegradation of the positive control substance.
Day		HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)		Degradation^1) (%)
		Blank (mean)	Positive control
1		49.72	50.00	0.00	0.0	0.0		0
4		47.09	27.73	19.36	21.3	21.3		25
6		49.53	39.52	10.01	11.0	32.3		38
8		49.24	41.63	7.61	8.4	40.7		47
11		48.47	41.13	7.34	8.1	48.7		57
13		48.86	46.40	2.46	2.7	51.5		60
15		48.30	43.46	4.84	5.3	56.8		66
20		46.92	43.40	3.52	3.9	60.6		71
25		43.86	41.25	2.61	2.9	63.5		74
29		43.17	41.43	1.74	1.9	65.4		76
29		43.91	46.45	0.00	0.0	65.4		76
29		47.32	47.07	0.25	0.3	65.7		77
^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.7 mg CO2/2l
Note: Day 14, calculated degradation : 63%

Table 3     CO2production and percentage biodegradation of the test substance (bottle A).
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Degradation^1) (%)
		Blank (mean)	bottle A
	1	49.72	50.50	0.00	0.0	0.0	0
	4	47.09	40.91	6.18	6.8	6.8	8
	6	49.53	42.48	7.05	7.8	14.6	17
	8	49.24	42.59	6.65	7.3	21.9	25
	11	48.47	40.13	8.34	9.2	31.0	36
	13	48.86	44.40	4.46	4.9	35.9	41
	15	48.30	45.65	2.65	2.9	38.9	45
	20	46.92	44.07	2.85	3.1	42.0	48
	25	43.86	41.02	2.84	3.1	45.1	52
	29	43.17	41.68	1.49	1.6	46.8	54
	29	43.91	42.18	1.73	1.9	48.6	56
	29	47.32	48.98	0.00	0.0	48.6	56
	^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 86.9 mg CO2/2l

Table 4     CO2production and percentage biodegradation of the test substance (bottle B).
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Degradation^1) (%)
		Blank (mean)	bottle B
	1	49.72	50.55	0.00	0.0	0.0	0
	4	47.09	39.44	7.65	8.4	8.4	10
	6	49.53	41.64	7.89	8.7	17.1	20
	8	49.24	42.50	6.74	7.4	24.5	28
	11	48.47	40.02	8.45	9.3	33.8	39
	13	48.86	45.01	3.85	4.2	38.0	43
	15	48.30	46.03	2.27	2.5	40.5	46
	20	46.92	45.55	1.37	1.5	42.0	48
	25	43.86	40.04	3.82	4.2	46.2	53
	29	43.17	43.78	0.00	0.0	46.2	53
	29	43.91	43.06	0.84	0.9	47.2	54
	29	47.32	47.00	0.32	0.4	47.5	54
	^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 87.6 mg CO2/2l

Table 5     CO2production and percentage biodegradation of the toxicity control.
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Degradation^1) (%)
		Blank (mean)	toxicity control
	1	49.72	49.07	0.65	0.7	0.7	0
	4	47.09	40.87	6.22	6.8	7.6	4
	6	49.53	38.20	11.33	12.5	20.0	12
	8	49.24	33.72	15.52	17.1	37.1	21
	11	48.47	28.69	19.78	21.8	58.8	34
	13	48.86	40.29	8.57	9.4	68.3	39
	15	48.30	38.67	9.63	10.6	78.9	45
	20	46.92	35.77	11.15	12.3	91.1	53
	25	43.86	34.33	9.53	10.5	101.6	59
	29	43.17	36.48	6.69	7.4	109.0	63
	29	43.91	45.02	0.00	0.0	109.0	63
	29	47.32	48.90	0.00	0.0	109.0	63
	^1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test substance
	and positive control: 173.3 mg CO2/2l
	ThCO2test substance: 87.6 mg CO2/2l
	ThCO2sodium acetate: 85.7 mg CO2/2l
	Note: Day 14, calculated degradation : 42%

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

In a ready biodegradability test with Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol (OECD 301B), 55% biodegradation was observed after 28 days. According to the criteria (60% biodegradation, within 10-day window) for ready biodegradability, Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is not ready biodegradable.

Executive summary:

In a ready biodegradability test with Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol (OECD 301B), 55% biodegradation was observed after 28 days. According to the criteria (60% biodegradation, within 10-day window) for ready biodegradability, Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is not ready biodegradable. The low water solubility of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol (< 1 mg/L), may have reduced the bioavailability of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol in the OECD 301B test, and as a consequence, reduced the rate of biodegradation observed in this test. This may have led to a failure of meeting the ready biodegradability criteria (i.e. 60% biodegradation, within 10-day window).

High levels (55%) of biodegradation were observed in OECD 301B test despite the low water solubility of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol.

The data do demonstrate that Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is ultimately biodegradable. The observed level of 55% biodegradation after 28 days implies that Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is completely mineralized to CO₂(with remainder incorporated into cellular material). Levels of >50% mineralization are typical for complete mineralization of substances with low water solubility.

The 28-day period can be extended, especially relevant for substances with low water solubility. The test (OECD301B) with Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was terminated after 28 days. Extrapolation of the curve indicates that the 60% level would have been reached after 30-32 days.

It is therefore concluded that Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol should be considered as readily biodegradable.

 

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

30 November, 2011 - 29 December, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999).

Qualifier:

according to guideline

Guideline:

other: ISO Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995).

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (33 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.
- Pretreatment: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Concentration of sludge: 1% final volume
- Initial cell/biomass concentration: The concentration of suspended solids was 4.1 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock solutions of A)8.50 g KH2PO4
mineral components 21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

Mineral medium 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

- Test temperature: Continuously measured in a vessel with Milli-RO water in the same room.
- pH: Measured at the start of the test and on the 28th day.
- pH adjusted: yes
- Aeration of dilution water: During the test period the test media were aerated and stirred continuously.
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration: Test suspension: containing test substance, silica gel and inoculum (2 bottles). Inoculum blank: containing only inoculum, silica gel (2 bottles). Positive control: containing reference substance, silica gel and inoculum (1 bottle). Toxicity control: containing test substance, silica gel and reference substance and inoculum (1 bottle).
- Preparation: Since Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was poorly soluble in water, weighed amounts (test substance bottle A and B: 29.9 mg and toxicity control bottle: 29.5 mg) were adsorbed to 100 mg granular silica gel (Sigma-Aldrich, Steinheim, Germany). Following adsorption to the silica gel the mixtures were dispersed in 300 ml of Milli-RO water. The test substance/silica gel mixture were strongly mixed for 15 minutes of megnetic stirring and thereafter transferred to the test vessels. Each of the dispersions was subsequently made up to final volume of 2 litres with test medium containing microbial organisms and mineral components. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.
At the start of the test (day 0) test and reference substance were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
-Method used to create aerobic conditions: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Measuring equipment: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul), Merck, Darmstadt, Germany).
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency/method: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the
28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used
as pH-indicator. On the 28th day, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The final titration was made on day 29.
- Sample storage before analysis: The bottles were aerated overnight to drive off CO2 present in the test suspension.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum and silica gel
- Toxicity control: containing test substance, reference substance, inoculum and silica gel

Reference substance:

other: Sodium acetate

Test performance:

Theoretical CO2 production:
Because the theoretical calculation of the CO2 production was not possible a sample of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was taken for TOC analysis.

Biodegradation:
All data are in the field 'Any other information on results incl. tables'. The results of CO2 production and biodegradation in each test bottle are listed in Tables 1-5.

The relative biodegradation values calculated from the measurements performed during the test period revealed 50 and 51% biodegradation of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control more than 25% biodegradation occurred within 14 days (52%, based on ThCO2).
Therefore, the test substance was assumed not to inhibit microbial activity.

Value:

ca. 51

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance):
3 % degradation after 2 d
12 % degradation after 5 d
20 % degradation after 7 d
32 % degradation after 9 d
42 % degradation after 14 d
47 % degradation after 19 d
49 % degradation after 23 d
49 % degradation after 27 d
49 % degradation after 29 d
51 % degradation after 29 d

Results with reference substance:

Points of degradation plot (reference substance):
11 % degradation after 2 d
39 % degradation after 5 d
51 % degradation after 7 d
60 % degradation after 9 d
70 % degradation after 14 d

Table1     CO2production in the blank.
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)
		Ba(OH)2^1)	Blank (mean)
	2	50.31	47.12	3.20	3.5	3.5
	5	50.57	46.49	4.08	4.5	8.0
	7	51.15	45.91	5.25	5.8	13.8
	9	49.87	45.85	4.02	4.4	18.2
	14	49.14	42.22	6.92	7.6	25.8
	19	49.06	41.75	7.31	8.0	33.8
	23	49.62	42.82	6.80	7.5	41.3
	27	50.31	42.06	8.25	9.1	50.4
	29	50.37	43.65	6.71	7.4	57.8
	29	48.39	46.48	1.91	2.1	59.9
	29	49.90	49.24	0.66	0.7	60.6
	^1): "Strength" of untreated 0.0125 M Ba(OH)2solution

Table 2    CO2production and percentage biodegradation of the positive control substance.
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
		Blank (mean)	Positive control
	2	47.12	38.72	8.40	9.2	9.2	11
	5	46.49	24.33	22.16	24.4	33.6	39
	7	45.91	36.65	9.26	10.2	43.8	51
	9	45.85	38.65	7.20	7.9	51.7	60
	14	42.22	34.62	7.60	8.4	60.1	70
	^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.8 mg CO2/2l

Table 3   CO2production and percentage biodegradation of the test substance (bottle A).
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
		Blank (mean)	bottle A
	2	47.12	45.21	1.90	2.1	2.1	2
	5	46.49	38.86	7.63	8.4	10.5	12
	7	45.91	38.58	7.33	8.1	18.5	21
	9	45.85	35.81	10.04	11.0	29.6	34
	14	42.22	34.96	7.26	8.0	37.6	43
	19	41.75	38.94	2.81	3.1	40.7	47
	23	42.82	41.47	1.35	1.5	42.1	49
	27	42.06	41.56	0.50	0.6	42.7	49
	29	43.65	44.10	0.00	0.0	42.7	49
	29	46.48	45.88	0.60	0.7	43.4	50
	29	49.24	48.93	0.31	0.3	43.7	50
	^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 86.7 mg CO2/2l

Table 4    CO2production and percentage biodegradation of the test substance (bottle B).
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
		Blank (mean)	bottle B
	2	47.12	45.05	2.07	2.3	2.3	3
	5	46.49	40.25	6.24	6.9	9.1	11
	7	45.91	39.16	6.75	7.4	16.6	19
	9	45.85	37.67	8.18	9.0	25.5	29
	14	42.22	33.19	9.03	9.9	35.5	41
	19	41.75	37.53	4.22	4.6	40.1	46
	23	42.82	41.21	1.61	1.8	41.9	48
	27	42.06	42.21	0.00	0.0	41.9	48
	29	43.65	44.26	0.00	0.0	41.9	48
	29	46.48	44.62	1.86	2.0	43.9	51
	29	49.24	49.37	0.00	0.0	43.9	51
	^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 86.7 mg CO2/2l

Table 5    CO2production and percentage biodegradation of the toxicity control.
	Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
		Blank (mean)	toxicity control
	2	47.12	36.83	10.29	11.3	11.3	7
	5	46.49	28.75	17.74	19.5	30.8	18
	7	45.91	32.71	13.20	14.5	45.3	26
	9	45.85	26.87	18.98	20.9	66.2	39
	14	42.22	20.74	21.48	23.6	89.8	52
	^1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test substance and    positive control: 171.4 mg CO2/2l (ThCO2test substance: 85.6 mg CO2/2l + ThCO2sodium acetate: 85.8 mg CO2/2l)

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

In a an enhanced ready biodegradability test with Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol (OECD 301B), 51% biodegradation was observed after 28 days. According to the criteria (60% biodegradation, within 10-day window) for ready biodegradability, Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is not ready biodegradable.

Executive summary:

An enhanced ready biodegradability test was performed with Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol (OECD 301B). In this test Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was coated onto silica gel through direct addition in an attempt to increase the bioavailability of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol in the ready biodegradability test. This test gave similar results (50-55% biodegradation after 28 days) compared to the standard test, and ready biodegradability (according the criteria) could not be concluded.

High levels (51%) of biodegradation were observed in OECD 301B test despite the low water solubility of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol.

The data do demonstrate that Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is ultimately biodegradable. The observed level of 51% biodegradation after 28 days implies that Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol is completely mineralized to CO₂(with remainder incorporated into cellular material). Levels of >50% mineralization are typical for complete mineralization of substances with low water solubility.

The 28-day period can be extended, especially relevant for substances with low water solubility. The test (OECD301B) with Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol was terminated after 28 days. Extrapolation of the curve indicates that the 60% level would have been reached after 30-32 days.

It is therefore concluded that Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol should be considered as readily biodegradable.

 

Description of key information

Two readily biodegradation tests were performed according to OECD guidelines and GLP principles.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

In a ready biodegradability test with Radia 7853 (OECD 301B), 55% biodegradation was observed after 28 days. According to the criteria (60% biodegradation, within 10-day window) for ready biodegradability, Radia 7853 is not ready biodegradable. The low water solubility of Radia 7853 (< 1 mg/L), may have reduced the bioavailability of Radia 7853 in the OECD 301B test, and as a consequence, reduced the rate of biodegradation observed in this test. This may have led to a failure of meeting the ready biodegradability criteria (i.e. 60% biodegradation, within 10-day window).

 

An enhanced ready biodegradability test was performed with Radia 7853. In this test Radia 7853 was coated onto silica gel through direct addition in an attempt to increase the bioavailability of Radia 7853 in the ready biodegradability test. This test gave similar results (50-55% biodegradation after 28 days) compared to the standard test, and ready biodegradability (according the criteria) could not be concluded.

High levels (55%) of biodegradation were observed in OECD 301B test despite the low water solubility of Radia 7853.

The data do demonstrate that Radia 7853 is ultimately biodegradable. The observed level of 55% biodegradation after 28 days implies that Radia 7853 is completely mineralized to CO₂(with remainder incorporated into cellular material). Levels of >50% mineralization are typical for complete mineralization of substances with low water solubility.

The 28-day period can be extended, especially relevant for substances with low water solubility. The test (OECD301B) with Radia 7853 was terminated after 28 days. Extrapolation of the curve indicates that the 60% level would have been reached after 30-32 days.

It is therefore concluded that Radia 7853 should be considered as readily biodegradable.