2,2-bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

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:

12 August 2015 to 10 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see Any other information

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see Any other information

Qualifier:

according to guideline

Guideline:

ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)

Deviations:

yes

Remarks:

see Any other information

Qualifier:

according to guideline

Guideline:

other: ISO International 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).

Deviations:

yes

Remarks:

see Any other information

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Source: 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.
Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 4.5 g/l in the concentrated sludge. Before use, the sludge was allowed to settle (47 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.
Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Duration of test (contact time):

28 d

Initial conc.:

36 - 36.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test procedure and conditions
Test duration: 28 days (last CO2 measurement on day 29). During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre glass brown coloured bottles.
Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO 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.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm): 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).
Illumination: The test media were excluded from light.

Preparation of bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles:
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test item, reference substance and inoculum (1 bottle).
Preparation: 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.
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.

Determination of CO2
Experimental CO2 production: 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® ampoule), Merck, Darmstadt, Germany).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of 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 day 28, 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 bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula.

Measurements and recording
pH: At the start of the test (day 0) and on day 28, before addition of concentrated HCl.
Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Reference substance:

acetic acid, sodium salt

Test performance:

Acceptability of the test
1. The positive control substance was biodegraded by at least 60% (64%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤4%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (44.8 mg CO2 per 2 litres of medium, corresponding to 22.4 mg CO2/l).
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/l).
Since all criteria for acceptability of the test were met, this study was considered to be valid.

Parameter:

% degradation (CO2 evolution)

Value:

4 - 6

Sampling time:

28 d

Details on results:

The ThCO2 of the test substance was calculated to be 2.46 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of the test substance (4% and 6%, based on ThCO2).
Significant biodegradation was observed in the toxicity control (21% in 14 days) and almost none in the test vessels (4-6% in 28 days). Therefore, it was concluded that the test substance is unlikely to be toxic. In case of toxicity limited or no biodegradation should have been observed in the toxicity control. Although, the biodegradation was just below the criteria set in the protocol the test substance was determined not to inhibit the microbial activity.
The temperature recorded in a vessel with water in the same room varied between 22 and 23°C.

Results with reference substance:

The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
The positive control substance was biodegraded by at least 60% (64%) within 14 days.

pH values of different test media

Test medium:	At the start of the test:	On day 28:
Blank control (A)	7.7 →^17.6	7.7
Blank control (B)	7.7 →^17.6	7.7
Positive control	7.7 →^17.6	8.0
Test substance (A)	7.7 →^17.6	7.7
Test substance (B)	7.7 →^17.6	7.7
Toxicity control	7.7 →^17.6	8.0

¹: Adjusted using 1 M HCI

 

Notes: Except for the percentages biodegradation, all calculations are performed without rounding off. Produced CO₂: negative values are expressed as 0.00 ml HCI.

 

HCI titrated in duplicate blank bottles

Day	HCI (0.05 N) titrated (ml)
	Blank A	Blank B	Mean Value
2 5 7 9 14 19 23 27 29 29 29	47.14 46.35 46.46 46.55 46.18 46.63 45.93 46.03 45.41 48.02 48.28	46.80 44.94 46.21 46.55 45.06 43.41 43.46 44.32 45.53 46.55 49.61	46.97 45.65 46.34 46.55 45.62 45,02 44.70 45.18 45.47 47.29 48.95

 

HCI titrated in Ba(OH)₂solution (background bottles)

Day	HCI (0.05 N) titrated (ml)
	Bottle A	Bottle B	Mean Value
2 5 7 9 14 19 23 27 29 29 29	49.73 49.64 50.00 50.00 51.25 50.63 50.50 50.09 50.27 49.05 49.80	49.71 50.51 48.85 50.99 48.98 49.90 50.75 47.72 49.13 50.00 49.41	49.72 50.08 49.43 50.50 50.12 50.27 50.63 48.91 49.70 49.53 49.61

 

CO₂production in the blank

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)
	Ba(OH)2^1)	Blank (mean)
2 5 7 9 14 19 23 27 29 29 29	49.72 50.08 49.43 50.50 50.12 50.27 50.63 48.91 49.70 49.53 49.61	46.97 45.65 46.34 46.55 45.62 45.02 44.70 45.18 45.47 47.29 48.95	2.75 4.43 3.09 3.95 4.49 5.25 5.93 3.73 4.23 2.24 0.66	3.0 4.9 3.4 4.3 4.9 5.8 6.5 4.1 4.7 2.5 0.7	3.0 7.9 11.3 15.6 20.6 26.4 32.9 37.0 41.6 44.1 44.8

¹⁾“Strength” of untreated 0.0125 M Ba(OH)₂solution

 

CO₂production and percentage biodegradation of the positive control substance

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Positive control
2 5 7 9 14	46.97 45.65 46.34 46.55 45.62	44.24 22.79 37.46 40.07 36.75	2.73 22.86 8.88 6.48 8.87	3.0 25.1 9.8 7.1 9.8	3.0 28.1 37.9 45.0 54.8	3 33 44 52 64

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of sodium acetate: 86.0 mg CO₂/2l

 

CO₂production and percentage biodegradation of the test item (bottle A)

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Bottle A
2 5 7 9 14 19 23 27 29 29 29	46.97 45.65 46.34 46.55 45.62 45.02 44.70 45.18 45.47 47.29 48.95	47.01 46.81 46.91 46.59 45.08 45.51 44.27 45.23 44.88 47.05 47.53	0.00 0.00 0.00 0.00 0.54 0.00 0.42 0.00 0.59 0.23 1.42	0.0 0.0 0.0 0.0 0.6 0.0 0.5 0.0 0.6 0.3 1.6	0.0 0.0 0.0 0.0 0.6 0.6 1.1 1.1 1.7 2.0 3.5	0 0 0 0 1 1 1 1 2 2 4

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 89.8 mg CO₂/2l

 

CO₂produced and percentage biodegradation of the test item (bottle B)

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Bottle B
2 5 7 9 14 19 23 27 29 29 29	46.97 45.65 46.34 46.55 45.62 45.02 44.70 45.18 45.47 47.29 48.95	47.67 44.33 47.35 45.98 44.83 45.02 44.34 43.92 45.45 46.77 50.00	0.00 1.32 0.00 0.57 0.79 0.00 0.35 1.26 0.02 0.51 0.00	0.0 1.4 0.0 0.6 0.9 0.0 0.4 1.4 0.0 0.6 0.0	0.0 1.4 1.4 2.1 2.9 2.9 3.3 4.7 4.7 5.3 5.3	0 2 2 2 3 3 4 5 5 6 6

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 90.8 mg CO²/2l

 

COproduced and percentage biodegradation of the toxicity control

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Toxicity control
2 5 7 9 14	46.97 45.65 46.34 46.55 45.62	40.78 38.50 36.82 42.60 39.76	6.19 7.15 9.52 3.95 5.86	6.8 7.9 10.5 4.3 6.4	6.8 14.7 25.1 29.5 35.9	4 8 14 17 21

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the sum of the ThCO₂of the test substance and positive control: 174.6 mg CO₂/2l (ThCO₂test substance: 88.6 mg CO₂/2l + ThCO₂sodium acetate: 86.0 CO₂/2l)

 

Comparison of biodegradation of the test item in bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆ A-B^1)
2 5 7 9 14 19 23 27 29 29 29	0 0 0 0 1 1 1 1 2 2 4	0 2 2 2 3 3 4 5 5 6 6	0 1 1 1 2 2 3 3 4 4 5	0 2 2 2 2 2 3 4 3 4 2

¹⁾Absolute difference in biodegradation between bottles A and B

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate].

 

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C, ISO 9439, 1999 and ISO 10634, 1995.

 

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off white pellets with a purity of 97.8%. The test item was tested in duplicate at a target concentration of 18 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was calculated to be 2.46 mg CO2/mg.

 

The study consisted of six bottles:

- 2 inoculum blanks (no test item),

- 2 test bottles (test substance),

- 1 positive control (sodium acetate) and

- 1 toxicity control (test substance plus sodium acetate).

 

Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test medium. Test duration was 28 days (last CO2-measurement on day 29).

 

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of the test substance (4% and 6%, based on ThCO2).

 

Although the biodegradation in the toxicity control was just below the criteria for toxicity set in the protocol it was concluded that the test substance did not inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was designated as not readily biodegradable.

Description of key information

Key value determined in GLP accredited laboratory to OECD Guideline 301 B and EC Method C.4-C.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate].

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off white pellets with a purity of 97.8%. The test item was tested in duplicate at a target concentration of 18 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was calculated to be 2.46 mg CO2/mg.

The study consisted of six bottles:

- 2 inoculum blanks (no test item),

- 2 test bottles (test substance),

- 1 positive control (sodium acetate) and

- 1 toxicity control (test substance plus sodium acetate).

Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test medium. Test duration was 28 days (last CO2-measurement on day 29).

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of the test substance (4% and 6%, based on ThCO2).

Although the biodegradation in the toxicity control was just below the criteria for toxicity set in the protocol it was concluded that the test substance did not inhibit microbial activity. 

In conclusion, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was designated as not readily biodegradable.