1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

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:

weight of evidence

Study period:

From 07 September to 06 October 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 301B (1992), EU Method C.4-C and US EPA Guideline OPPTS 835.3110 Paragraph (m) with GLP statement. All validity criteria were fulfilled. This study is reliable without restriction.

Qualifier:

according to guideline

Guideline:

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

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)

Version / remarks:

Paragraph (m)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Stability under test conditions: stable
- Storage condition of test material: in the dark at approximately 4°C under nitrogen

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aeration stage of Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

-Method of preparation of the inoculum: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium and then was maintained on continuous aeration in the laboratory at a temperature of 21ºC and was used on the day of collection. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a washed sample (100 ml) of the activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. The filter paper was then dried in an oven at approximately 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 was equal to 2.8 g/l prior to use.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l.

Duration of test (contact time):

28 d

Initial conc.:

13.2 mg/L

Based on:

test mat.

Initial conc.:

10 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:

a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The study was carried out in a temperature controlled room at 21°C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 32.1 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing selfindicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

TEST CONDITIONS
- Composition of medium: medium as recommended in the OECD 301B guideline: To 1 liter (final volume) of purified water (Reverse osmosis and deionised water) was added the following volumes of solutions a-d: 10 ml of solution a and 1ml of solutions b, c & d. Compositions of solutions a to d: solution a: KH2PO4 8.5 g/l, K2HPO4 21.75 g/l, Na2HPO4.2H2O 33.40 g/l, NH4Cl 0.50 g/l, pH7.4. Solution b: CaCl2: 27.50 g/l. Solution c: MgSO4.7H20 22.50 g/l. Solution d: FeCl3.6H20: 0.25 g/l.
- Solubilising agent (type and concentration if used): none
- Test temperature: 21°C
- pH: 7.5 to 7.8
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/l
- Continuous darkness: yes


SAMPLING
- Sampling frequency: Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29. The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12 and 18 were stored at approximately -20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly
increase during this time and therefore additional analyses were considered to be unnecessary.
On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.


CONTROL AND BLANK SYSTEM (see above)
- Inoculum blank: yes
- Procedure control: yes (Na benzoate in inoculated culture medium)
- Toxicity control: yes (test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only)).

Reference substance:

benzoic acid, sodium salt

Remarks:

Sigma lot N°043K0107

Preliminary study:

A study to determine the General Physico-Chemical properties of the test material gave a water solubility value of less than or equal to 9.3 x 10-5 g/l at 20ºC±0.5ºC. Therefore the following preliminary solubility work was conducted to ascertain the method that would give the best testable dispersion: (i) Direct addition (this formed a cloudy dispersion with oily globules of test material on the surface) (ii) Ultrasonication for 30 min (this formed a slightly cloudy dispersion with a large globule of test material on the surface) (iii) High shear mixing (7500 rpm for 15 min); this formed a cloudy dispersion with no undissolved test material visible (iv) High shear mixing with the aid of silica gel; this formed a cloudy dispersion with no undissolved test material visible.
From this preliminary solubility work it was concluded that the best testable dispersion of the test material was obtained with the aid of high shear mixing with and without the use of silica gel. A volatility check was then performed on the test material by passing a steady stream of compressed air over a known weight of test material for 3 hours as high shear mixing cannot be performed on volatile test materials. This indicated a 1% loss of test material over this time so the test material was considered not to be volatile. As there was no difference between high shear mixing the test material with and without silica gel it was considered appropriate not to use silica gel in the test preparations, as it did not increase the dispersibility of the test material when subjected to high shear mixing. For the purpose of the study the test material was therefore dispersed directly in culture medium with the aid of high shear mixing.

Test performance:

No unusual observations reported during the study

Key result

Parameter:

% degradation (CO2 evolution)

Value:

26

St. dev.:

0

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance): 0 % degradation after 3 d 0 % degradation after 6 d 2 % degradation after 10 d 9 % degradation after 14 d 12 % degradation after 16 d 13 % degradation after 20 d 14 % degradation after 22 d 15 % degradation after 24 d 26 % degradation after 28 d
(See Biodegradation curves in "attached backgroun material")

Results with reference substance:

Points of degradation plot (reference substance):
65 % degradation after 3 d
67 % degradation after 6 d
77 % degradation after 10 d
81 % degradation after 14 d
86 % degradation after 16 d
88 % degradation after 20 d
91 % degradation after 22 d
91 % degradation after 24 d
95 % degradation after 28 d
(See Biodegradation curves in "attached backgroun material")

None

Validity criteria fulfilled:

yes

Remarks:

See comments made in Overall Remarks (text to short here)

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction. A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

 

Methods.The test material, at a concentration of 10 mg C/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21°C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

Results.The test material attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to the OECD Guidelines without GLP statement to assess the biodegradability of the test substance using prolonged Closed Bottle tests. Validity criteria cannot be checked for this moment because raw data are not available in this report. However, the detail of methodology is well documented.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Deviations:

no

Principles of method if other than guideline:

The tests were modified to permit prolonged measurements (van Ginkel and Sroo, 1992).
The Closed Bottle tests have been inoculated with river water, unacclimated activated sludge, sludge from the SCAS unit and microorganisms from an enrichment culture.

GLP compliance:

no

Remarks:

This screening study was not performed under GLP as the intention was to investigate the fate of the registered substance using different test conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

other: river water, unacclimated activated sludge, sludge from the SCAS unit and microorganisms from an enrichment culture.

Details on inoculum:

The Closed Bottle tests have been inoculated with river water, unacclimated activated sludge, sludge from the SCAS unit and microorganisms from an enrichment culture.

- River water: River water was sampled from the Rhine near Heveadorp, The Netherlands. River water was aerated for 5 to 7 days before use. The particles were removed by sedimentation.
- Unacclimated activated sludge: Secondary activated sludge and primary settled sewage were obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The primary settled sewage was collected weekly and stored at -20°C until required. The activated sludge for the Closed Bottle test was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992). 150 mL of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit.
- Sludge from the SCAS unit: The SCAS test was performed in 150 ml SCAS units. At the start each SCAS unit was filled with 150 ml of activated sludge(2 g DW/L of suspended solids) and the aeration was started. After 23 hours the aeration was stopped and the sludge was allowed to settle for 45 minutes. After settling 100 ml of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 ml deionized water were added to the control unit, and 95 ml of primary settled sewage and 5 ml of a test substance stock suspension were added to the test unit. A stock suspension of the registered substance (EC 456-350-3)(1.0 g/L) and Tween 80 (1.0 g/L) in water was prepared by ultrasonic dispersion at 200 W for 5 minutes using a Vibra-cell. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test (three months). During this period a two Closed Bottle tests were inoculated with adapted sludge from the SCAS unit. The registered susbtance was administered in the bottles using a suspension of the registered substance and Tween 80.
- Microorganisms from an enrichment culture: The enrichment was cultured in a medium composed a mineral salt medium and silicone oil at a 9:1 volume ratio. The silicone oil contained 20.0 g/L of the registered substance (EC 456-350-3) giving a final concentration of the regsitered substance in the culture medium 2.0 g/L. The organism was cultured in a mineral medium. The medium was initially inoculated with 1 mL of activated sludge from a SCAS unit exposed to the registered substance for more than 100 days. When growth was observed, fresh medium with the same composition was inoculated with a few droplets. This procedure was repeated a few times. A Closed Bottle test was inoculated with microorganisms present in the aqueous phase of the enrichment culture.

Duration of test (contact time):

112 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Deionized water containing no more than 0.01 mg/L Cu was prepared in a water purification system. The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg Ca-Cl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Solubilising agent (type and concentration if used): The registered substance ( EC 456-350-3) is a poorly soluble substance in water. Administration of registered substance ( EC 456-350-3) was accomplished by dosing 0.04 g of registered substance ( EC 456-350-3) on 10 g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with alumiminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with registered substance ( EC 456-350-3) was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L.
- Test temperature: The temperature was measured and recorded with a thermo couple connected to a data logger. 23 +/- 1°C
- pH: The pH was measured using a Eutech Cyberscan pH11 pH meter (Eutech Instruments, Nijkerk, The Netherlands). The pH of the effluent of the SCAS units ranged from 6.0 to 7.5.
- pH adjusted: no
- Aeration of dilution water:
- Suspended solids concentration: The activated sludge (unacclimated and acclimated) was diluted to 2 mg DW/L in the mineral salts medium and river water were used undiluted spiked with mineral salts.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The tests were performed in 0.3L BOD bottles with glass stoppers.
- Number of culture flasks/concentration: 3 bottles containing only inoculum, and 3 bottles containing inoculum and test substance administered using silica gel.
- Method used to create aerobic conditions: no data
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands).
- Other: All bottles were completely filled without air bubbles.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 bottles containing only inoculum

Preliminary study:

None

Test performance:

The ThOD used to calculate the biodegradability of test substance was 3.0 mg/mg.

Parameter:

% degradation (O2 consumption)

Value:

20

Sampling time:

84 d

Remarks on result:

other: Silica gel (2.0 mg/L), sludge

Parameter:

% degradation (O2 consumption)

Value:

21

Sampling time:

112 d

Remarks on result:

other: Silica gel (2.0 mg/L), river water

Parameter:

% degradation (O2 consumption)

Value:

21

Sampling time:

84 d

Remarks on result:

other: Silica gel (2.0 mg/L), sludge from SCAS unit 6 weeks

Parameter:

% degradation (O2 consumption)

Value:

15

Sampling time:

28 d

Remarks on result:

other: Silica gel (2.0 mg/L), sludge from SCAS unit 13 weeks

Details on results:

The test substance did degrade approximately 20% in the prolonged Closed Bottle test using unadapted activated sludge, unacclimated river water, and acclimated sludge from the SCAS unit. Acclimatization only resulted in a decrease of the lag period. An enrichment culture was developed from activated sludge of the SCAS unit to ease analysis of the organic substances not susceptible to microbial degradation biodegradation. Microorganisms of the enrichment culture used as inoculums in a Closed bottle test also degrade the test substance for approximately 20%. 

See table 5.2.1/1 in "Any other information on results incl. tables" and graph in "Attached background material".

Results with reference substance:

Not applicable

Table 5.2.1/1: Summary of results obtained with the test substance in Closed Bottle tests

Method (initial concentration of the test substance) <inoculum>	Biodegradation (%) at
	Day 28	Day of the test between brackets
Silica gel (2.0 mg/L) <sludge>	8	20 (84)
Silica gel (2.0 mg/L) <river water>	3	21 (112)
Silica gel (2.0 mg/L) <sludge from SCAS unit 6 weeks>	18	21 (84)
Silica gel (2.0 mg/L) <sludge from SCAS unit 13 weeks>	15
Silica gel (2.0 mg/L) <enrichment culture>

Validity criteria fulfilled:

not specified

Remarks:

the raw data are not available

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance did degrade approximately 20% in the prolonged Closed Bottle test using unadapted activated sludge, unacclimated river water, acclimated sludge from the SCAS unit and microorganisms from enrichment culture. The test substance is considered not to be readily biodegradable.

Executive summary:

This study was performed, according to the OECD Guidelines without GLP statement, to assess the biodegradability of the test substance using prolonged Closed Bottle tests. The tests were modified to permit prolonged measurements, and have been inoculated with river water, unacclimated activated sludge (from the wastewater treatment plant, predominantly domestic sewage) , sludge from the SCAS unit and microorganisms from an enrichment culture.

The SCAS test was performed in 150 ml SCAS units. At the start each SCAS unit was filled with 150 ml of activated sludge (2 g DW/L of suspended solids) and the aeration was started. After 23 hours the aeration was stopped and the sludge was allowed to settle for 45 minutes. After settling 100 ml of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 ml deionized water were added to the control unit, and 95 ml of primary settled sewage and 5 ml of a test substance stock suspension were added to the test unit. A stock suspension of the test substance (1.0 g/L) and Tween 80 (1.0 g/L) in water was prepared by ultrasonic dispersion at 200 W for 5 minutes using a Vibra-cell. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test (three months). During this period a two Closed Bottle tests were inoculated with adapted sludge from the SCAS unit (after 6 and 13 weeks). The test substance was administered in the bottles using a suspension of test substance and Tween 80.

To obtain microorganisms from an enrichment culture, the enrichment was cultured in a medium composed a mineral salt medium and silicone oil at a 9:1 volume ratio. The silicone oil contained 20.0 g/L of test substance giving a final concentration of 2.0 g/L in the culture medium. The organism was cultured in a mineral medium. The medium was initially inoculated with 1 mL of activated sludge from a SCAS unit exposed to the test substance for more than 100 days. When growth was observed, fresh medium with the same composition was inoculated with a few droplets. This proceduere was repeated a few times. A Closed Bottle test was inoculated with microorganisms present in the aqueous phase of the enrichment culture.

In the Closed Bottles tests, the resulting concentration of test substance in the bottles was 2.0 mg/L.The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers.Use was made of 3 bottles containing only inoculum, and 3 bottles containing inoculum and test substance administered using silica gel. The activated sludge (unacclimated and acclimated) was diluted to 2 mg DW/L in the mineral salts medium and river water were used undiluted spiked with mineral salts. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at 23±1°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles with a special funnel.

The ThOD used to calculate the biodegradability of test substance was 3.0 mg/mg.

The test substance did degrade approximately 20% in the prolonged Closed Bottle test using unadapted activated sludge, unacclimated river water, and acclimated sludge from the SCAS unit. Acclimatization only resulted in a decrease of the lag period. An enrichment culture was developed from activated sludge of the SCAS unit to ease analysis of the organic substances not susceptible to microbial degradation biodegradation. Microorganisms of the enrichment culture used as inoculums in a Closed bottle test also degrade the test substance for approximately 20%. 

Molecules of the test substance are either degraded partial or only a few of the steroisomers are degraded completely. The other option is degradation of a part of the molecules. The biodegradation is probably not initiated by cleavage of the test substance. The most likely biodegradation pathway is oxidation of the alkyl chain from the far-ends.

In conclusion, the test substance is considered not to be readily biodegradable.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 December 2006 to 10 April 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study was performed according to Japanese national guideline, equivalent to OECD Guideline 301 C with GLP statement. Only basic data were provided in this report. Restrictions must be taken into account with this method.

Qualifier:

according to guideline

Guideline:

other: Japanese national guidelines: (Yakushokuhatsu N°1121002, Heisei 15.11.13 Seikyoku No.2, Kanpokihatsu N°031121002, November 21, 2003 ; the latest revision, November 20,2006).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

April 10, 2007

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Key result

Parameter:

% degradation (O2 consumption)

Value:

1

St. dev.:

1.7

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance): 1 % degradation after 28 d

None

Validity criteria fulfilled:

yes

Remarks:

1.) Biodegradability aniline is greater than 60% after 14 days. 2) The difference of extremes of replicate values of biodegradability at day 28 is less than 20 %. 3) The BOD in the control blank (bottle 2) is less than 18 mg O2 after 28 days.

Interpretation of results:

not readily biodegradable

Conclusions:

From the degradability results based on the BOD (average 1%) it is concluded that the test substance is not readily biodegradable, and is not transformed into any degradants.

Executive summary:

Introduction. Information on the biodegradability of the test material by microorganism was obtained using an assay in accordance with Japanese Biodegradability National Standards (<Biodegradability Test of Chemical Substances by Microorganisms> (Yakushokuhatsu N°1121002, Heisei 15.11.13 Seikyoku N°2, Kanpokihatsu N°031121002, November 21, 2003 ; the latest revision, November 20,2006).) and with OECD 301C test guideline.

Methods & Results. The activated sludge was provided by the Chemicals Evaluation and Research Institute of Japan. The culturing was performed at 25 °C. Concentration of suspended solid in the activated sludge was 3900 mg/L. The reference substance used was aniline. Test solutions were prepared as follows: 

Bottle 1: Aniline + activated sludge+basal medium.30 mg of aniline was added to the basalmedium, then activated sludge was added.

Bottle 2: Activated sludge + basal medium. Activated sludge was added to the basal medium

Bottles 3-5: Test substance + activated sludge + basal medium. 30 mg of the test substance was added to the basal medium, then activated sludge was added.

Bottle 6: Test substance + purified water. 30 mg of the test substance was added to 300 ml of purified water

 

A closed system oxygen consumption measuring apparatus was used. Cultivation was performed at 25°C for 28 days. The values (from three replicates) for degree of degradation based on the Biological Oxygen Demand were 0%, 0%, and 2% (mean 1%), whereas the degree of biodegradation of the reference substance (aniline) was greater than 60% after 14 days, indicating that the test conditions were valid.

 

Conclusions: The test substance is not readily biodegradable according to the Japanese Biodegradability National Standards and according to the OECD 301C test guideline.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-09-10 to 2012-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 301 F. All validity criteria were fulfilled.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

This study was not performed under GLP as the intention was to investigate, in depth, the fate of the registered substance in a R&D laboratory and to allay potential PBT concerns for the registered substance and its metabolites.

Specific details on test material used for the study:

- Water Solubility (25°): <0.5 μg/L. (measured)
- Vapor pressure: 7.5 x 10^-6 Pa at 25 °C (measured)
- Partition coefficient (logPow): 9.7 at 25 °C (measured)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge was collected from the wastewater treatment plant of Villette (canton Geneva, Switzerland) which predominantly treats municipal sewage.
- The sample of activated sewage sludge was maintained under continuous aeration for ~24h. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in mineral medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained by aeration with pure oxygen at room temperature. Determination of dry weight was made to inoculate final solution with 30 mg/L dry weight activated sludge.

Duration of test (contact time):

28 d

Initial conc.:

98 mg/L

Based on:

test mat.

Remarks:

Test 1

Initial conc.:

97 mg/L

Based on:

test mat.

Remarks:

Test 2

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was made up according to the OECD guideline and contained the following nutrients per litre of MilliQ: 85 mg of KH2PO4, 217.5 mg of K2HPO4, 334 mg of Na2HPO4.2H2O, 5 mg of NH4Cl, 22.5 mg of MgSO4.7H2O, 36.4 mg of CaCl2.2H2O, 0.25 mg of FeCl3.6H2O.
- Test temperature: 22 ºC
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Suspended solids concentration: All test vessels contained the prepared activated (30 mg/l) sludge inoculum in mineral medium.
- Continuous darkness: No

TEST SYSTEM
- Culturing apparatus: 250 mL bottles
- The bottles have an actual volume of 310 mL, and 100 mL of test solution was added to each bottle. The bottles were incubated at 22 ºC for up to 28 days. The samples were stirred for the duration of the study with a magnetically coupled stirrer.
- Number of culture flasks/concentration: 2
- Measuring equipment: WTW oxitopC calculates automatically the consumption of oxygen allowing it to be expressed as a % of THOD.
- The test was carried out in nominal 250mL bottles in diffused light, sealed by a sensor head/CO2 trap in a temperature controlled incubator. As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed by a NaOH trap causing a net reduction in gas pressure within the sample flask.

SAMPLING
- Sampling frequency: 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum blanks contained no test substance, to measure the sludge endogenous respiration, and to demonstrate there was no other carbon source in the medium and used to correct the relevant test vessels.
- Abiotic sterile control: Abiotic control contained test material at ~100 ppm, to demonstrate the stability of the test material in the mineral medium under test conditions. The test material was dispersed directly in culture medium. An amount of test material (9.6 mg) was dispersed in 100 mL of culture medium inoculated to give the test concentration of 96 mg/L with abiotic agent (NaN3).
- Toxicity control: Toxicity control contained sodium benzoate at ~100 ppm (97 mg/L) and test material at ~100 ppm (105 mg/L), to reveal a potential inhibitory effect of the test material on sludge.
- Positive control: Positive control contained sodium benzoate at ~100 ppm, to demonstrate the viability of the inoculum.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not applicable

Key result

Parameter:

% degradation (O2 consumption)

Value:

16

Sampling time:

28 d

Remarks on result:

other: mean degradation of test 1 and 2

Details on results:

The test material (test 1) attained 18 % degradation after 28 days. The test material (test 2) attained 14 % degradation after 28 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 301F.
The toxicity test attained 40% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Key result

Parameter:

ThOD

Value:

3.04 other: mgO2/mg substance

Results with reference substance:

Sodium Benzoate attained 93% degradation after 28 days thereby confirming test validity, the suitability of the inoculum and test conditions.

Table 5.2.1/2: Biodegradation values after 28 days   Samples 28 days biodegradation% Samples name Test 4 93 Reference Test 5 18 Test 1 Test 6 14 Test 2 Test 7 40 Toxicity Test 8 0 Abiotic

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material (test 1, test 2) attained respectively 18% and 14% degradation (16% mean degradation) after 28 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 301F. Nevertheless, sufficient mineralisation occurred that complete primary degradation of the parent structure is ensured within this time frame.

Executive summary:

This study was performed according to the OECD Guideline 301 F, to determine the biodegradability of the test substance by the Manometric Respirometry Test.

 

The test substance was exposed to activated sludge (30 mg/L) at a concentration of 100 ppm (98 mg/L and 97 mg/L in test 1 and 2, respectively) with culture medium in closed bottles at 22 °C for 28 days. The degradation of the test material was assessed by the measurement of oxygen consumption. Inoculum blank, abiotic control, toxicity control and positive controls were included in the test.

 

The test material (test 1) attained 18 % degradation after 28 days. The test material (test 2) attained 14 % degradation after 28 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 301F.

The toxicity test attained 40% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium Benzoate attained 93% degradation after 28 days thereby confirming test validity, the suitability of the inoculum and test conditions.

 

The test material attained 16% mean degradation after 28 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 301F. Nevertheless, sufficient mineralisation occurred that complete primary degradation of the parent structure is ensured within this time frame.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-05-04 to 2012-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 301 F. All validity criteria were fulfilled.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

This study was not performed under GLP as the intention was to investigate, in depth, the fate of the registered substance in a R&D laboratory and to allay potential PBT concerns for the registered substance and its metabolites.

Specific details on test material used for the study:

'- Water Solubility (25°): <0.5 μg/L at 25°C (measured)
- Vapor pressure: 7.5 x 10^-6 Pa at 25 °C (measured)
- Partition coefficient (logPow): 9.7 at 25 °C (measured)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge was collected from the wastewater treatment plant of Villette (canton Geneva, Switzerland) which predominantly treats municipal sewage.
- The sample of activated sewage sludge was maintained under continuous aeration for ~24h. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in mineral medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.

Duration of test (contact time):

84 d

Initial conc.:

100 other: ppm

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was made up according to the OECD guideline and contained the following nutrients per litre of MilliQ: 85 mg of KH2PO4, 217.5 mg of K2HPO4, 334 mg of Na2HPO4.2H2O, 5 mg of NH4Cl, 22.5 mg of MgSO4.7H2O, 36.4 mg of CaCl2.2H2O, 0.25 mg of FeCl3.6H2O.
- Test temperature: 22 ºC
- Suspended solids concentration: All test vessels contained the prepared activated (30 mg/l) sludge inoculum in mineral medium.
- Continuous darkness: No

TEST SYSTEM
- Culturing apparatus: 250 mL bottles
- The bottles have an actual volume of 310 mL, and 100 mL of test solution was added to each bottle. The bottles were incubated at 22 ºC for up to 28, 56 and 84 days. The samples were stirred for the duration of the study with a magnetically coupled stirrer.
- Number of culture flasks/concentration: Nine replicates of each set of vessels were prepared to allow for triplicate vessels to be sacrificed on three sample occasions (days 28, 56 and 84).
- Measuring equipment: WTW oxitopC calculates automatically the consumption of oxygen allowing it to be expressed as a % of THOD.
- The test was carried out in nominal 250mL bottles in diffused light, sealed by a sensor head/CO2 trap in a temperature controlled incubator. As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed by a NaOH trap causing a net reduction in gas pressure within the sample flask.
SAMPLING
- Sampling frequency: 28, 56 and 84 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum blanks contained no test substance, to measure the sludge endogenous respiration, and to demonstrate there was no other carbon source in the medium and used to correct the relevant test vessels.
- Positive control was directly observed on the test samples. As several test were conducted on the test substance, partial degradation demonstrate the viability of the inoculum.

Reference substance:

not specified

Preliminary study:

Not applicable

Key result

Parameter:

% degradation (O2 consumption)

Value:

43

Sampling time:

84 d

Details on results:

Manometric Respirometry: The test substance attained 30% degradation at the plateau, thereby confirming the inoculum viability as similar results to these were obtained in a previous study (DBO R Lsb06) where the reference criteria were met.

Key result

Parameter:

ThOD

Value:

3.04 other: mg O2/mg test substance

Table 5.2.1/1: Experimental design

 

Tests	Name	Planned mg		Real mg		Innoculated Mineral medium [L]
		Product	Ref	Product	Ref
1	Blank 1	-	-	-	-	0.1
2	Blank 2	-	-	-	-	0.1
3	Blank 3	-	-	-	-	0.1
4	Blank 1 / 28	-	-	-	-	0.1
5	TST 1/28	10	-	9.5	-	0.1
6	TST 2/28	10	-	11.2	-	0.1
7	TST 3/28	10	-	10.2	-	0.1
14	Blank 1 / 56	10	-	-	-	0.1
15	TST 1/56	10	-	9.1	-	0.1
16	TST 2/56	10	-	11.0	-	0.1
17	TST 3/56	10	-	10.3	-	0.1
24	Blank 1 / 84	10	-	-	-	0.1
25	TST 1/84	10	-	10.4	-	0.1
26	TST 2/84	10	-	12.1	-	0.1
27	TST 3/84	10	-	9.7	-	0.1

For a molecule C_cH_hCl_clN_nNa_naO_oP_pS_swith a molecular weight MW, the ThOD is:

DThONH₃= (16[2c+1/2(h-cl-3n)+3s+5/2p+1/2na-o]mg/mg) / (MW)

For the test substance, C₂₅H₄₆O₁S₁ yields a molecular weight of 394.70 g/mol

ThOD = 3.040 mgO2/mg substance.

 

Table 5.2.1/2: Ultimate Biodegradation values for test compound for three incubation times

 

Samples	28 days biodegradation%	56 days biodegradation%	84 days biodegradation%
Test 5	12*	-	-
Test 6	5*	-	-
Test 7	1*	-	-
Mean	6	-	-
Test 15	3	28*	-
Test 16	19	31*	-
Test 17	4	25*	-
Mean	8.7	28.0	-
Test 25	13	40	47*
Test 26	10	28	35*
Test 27	12	32	46*
Mean	11.7	33.3	42.7

*: sacrificed for analysis of metabolites

 

Analytical Results

After 28, 56 and 84 days of incubation, the manometric sensor head with the CO2 trap was removed from each vessel head. The vessel content was transferred into an extraction funnel. The reaction vessel, magnetic stirrer and vessel head were rinsed with methylene chloride (50 ml), and then the reaction mixture was extracted with the rinse solvent. This rinse and extraction procedure was repeated another two times. The pooled extracts were concentrated to ca. 2 ml under partial vacuum (100 mbar) at a maximum bath temperature of 40°C (Rotavapor). GC injection (parameters below) showed that only trace amounts of volatile metabolites (eluting before Delta Damascone) are present. Delta Damascone is observed as the main peak, mainly as a result of extensive thermal degradation in the injector of the metabolites and remaining test compound. The extract was further concentrated (40°C, ca. 10 mbar). Note that the aim of the present study wasn’t to achieve constant weight in order to determine the exact mass of the extract. A sample of the concentrated extract was diluted with ca. 20 parts of toluene in a GC vial and treated with excess MSTFA (80°C). Progress of the derivatisation was monitored by GC using a methylsilicone column. After 1 h no further change in composition occurred, indicating complete derivatisation. GC analysis was done using the instrument parameters below.

 

Typical gas chromatograph parameters:

• GC: 6850 Series II with FID detector, 6850 Autosampler

• Column: J&W Scientific DB-1

• Length: 10 meters, 0.10 millimeter inside diameter, 10 micrometer film thickness

• Method: split injection: >100/1 split ratio

• 0.20 microliters per injection

• Column flow: 0.9 milliliters hydrogen per minute

• Air flow: 450 milliliters per minute

• Inlet temperature: 250 degrees Centigrade

• Detector temperature: 320 degrees Centigrade

• Initial temperature = 60 degrees Centigrade, 50 degrees Centigrade per minute ramp rate, final temperature = 310 degrees Centigrade, Final time = 1 minute.

 

In addition to a small percentage of remaining parent, the metabolites below were detected over the study period. An internal standard was used to be sure about the percentage obtained during the GC analysis. All metabolites were characterized by comparison (GC retention time; MS) with authentic material:

• 2-mercapto acetic acid bis TMS derivative (UMBM-2012-01-899)

• Delta damascone (item#937856 lot 1000839061)

• DD mercapto acetic acid TMS ester (UMBM-2012-01-909)

• DD mercapto propionic acid TMS ester (UMBM-2012-01-913)

 

Table 5.2.1/3: Metabolite and parent concentration as a percentage at day 28, 56 and 84

 

Test ref.	Days	A [%]	B [%]	C [%]	D [%]	E [%]	F [%]	Total recovery
RNE1600/TST BUT 1.28 DBO R LSB 060 T05	28	0.12	8.05	0.13	10.16	4.6	58.09	73.56
RNE1601 / TST BUT 2.28 DBO R LSB 060 T06	28	0.12	1.62	0.13	0.81	1.05	61.74
RNE1602 / TST BUT 3.28 DBO R LSB 060 T07	28	0.12	0.7	0.07	0	0	64.94
average	28	0.12	3.46	0.11	5.45	2.83	61.59
RNE1616 / TST BUT 1.56 DBO R LSB 060 T15	56	0	12.46	7.82	0.49	1.8	49.98	66.26
RNE1617 / TST BUT 2.56 DBO R LSB 060 T16	56	0	9.15	9.66	21.07	8.99	16.53
RNE1618 / TST BUT 3.56 DBO R LSB 060 T17	56	0	11.86	10.35	4.66	2.45	31.5
average	56	0	11.16	9.28	8.74	4.41	32.67
RNE1629 / TST BUT 1.56 DBO R LSB 060 T25	84	0.19	6.19	33.34	4.4	1.7	23.09	64.19
RNE1630 / TST BUT 2.56 DBO R LSB 060 T26	84	1.36	10.33	11.38	25.68	5.7	6.64
RNE1631 / TST BUT 3.56 DBO R LSB 060 T27	84	0.13	4.5	32.03	13.03	3.53	9.35
average	84	0.56	7.01	25.58	14.37	3.64	13.03

  

Percentages are calculated using an internal standard and are percentage of the compound in the extracted residue.

The quantification is based on the GC peak area. It should be noted that the analytical mass balance is between 64 to 74% based on recovery from extracted residue. Most of the remaining unrecovered substance will be due to the mineralisation of the substance while some further loss could potentially happen during the extraction and evaporation process despite the care taken. While a complete mineralisation and analysed residue mass balance would be difficult to perform, it can be assumed that the majority of the mass balance has been accounted for.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Based on the results, the test substance is not considered readily biodegradable but the study indicates total primary biodegradation of the substance and partial mineralisation. Therefore the substance can be considered as inherently biodegradable, not fulfilling specific criteria and not persistent. The main biodegradation products have been clearly identified by independent synthesis. The two main degradation products are Delta Damascone-mercapto acetic acid (DD-ma) and Delta Damascone-mercapto propionic acid (DD-mp).

Executive summary:

The degradation test was performed using the Manometry Respirometry Method outlined in OECD 301F, but with modifications of the standard guideline to determine and quantify the degradation products of the test substance in order to identify and propose a degradation pathway. This study was carried out as a supplementary test to a standard OECD 301F (DBO R Lsb063) including modifications to allow analysis of metabolites at different incubation times over the study period.

 

The test substance was exposed to activated sludge (30 mg/L) at a concentration of 10 mg (9.5, 11.2 & 10.2 mg/L in test 1 (28 days), 9.1, 11.0 & 10.3 mg/L in test 2 (56 days), 10.4, 12.1 & 9.7 mg/L in test 3 (84 days)) with culture medium in closed bottles at 22 °C for 28/56/84 days. The degradation of the test material was assessed by the measurement of oxygen consumption.

 

In the test assessing biodegradability using OECD 301F with the design modified to prolong the duration to 84 days and to incorporate a full analysis of metabolites at three time points, the following mineralisation values were determined:

 

Samples	Mean biodegradation after 28 days (%)	Mean biodegradation after 56 days (%)	Mean biodegradation after 84 days (%)
Test 5, 6, 7	6*	-	-
Test 15, 16, 17	9	28*	-
Test 25, 26, 27	12	33	43*

*: sacrificed and analyzed for metabolites

 

Mean degradation was 43%, min 35%; max 47% after 84 days incubation.

 

The test substance is not considered readily biodegradable but the study indicates total primary biodegradation of the substance and partial mineralisation. Therefore the substance can be considered as inherently biodegradable, not fulfilling specific criteria and not persistent. The main biodegradation products have been clearly identified by independent synthesis. The two main degradation products were Delta Damascone-mercapto acetic acid (DD-ma) and Delta Damascone-mercapto propionic acid (DD-mp).

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-03-15 to 2012-04-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 301 F. All validity criteria were not fulfilled

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

All respirometers maintained until day 41 of the study; no OECD reference substance; biodegradation determined on days 28 and 41, thus rate of degradation could not be determined; validity criteria for pH at the end of the study not considered

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

This study was not performed under GLP as the intention was to investigate, in depth, the fate of the registered substance in a R&D laboratory and to allay potential PBT concerns for the registered substance and its metabolites.

Specific details on test material used for the study:

- Physical state and appearance: Colorless liquid
- Molecular formula: C17H28O3S
- Molecular weight: 312,47 g/mol
- Water Solubility (25°): 5.065 mg/L (Epiweb 4.0)
- Vapor pressure: 2.52E-005 Pa (Epiweb 4.0)
- Partition coefficient (logPow): 4.34 (Epiweb 4.0)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Sludge was collected from the municipal wastewater treatment plant of Villette (canton Geneva, Switzerland) which predominantly treats municipal sewage.
- The sample of activated sewage sludge was maintained under continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Hettich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained with pure oxygen at room temperature at least 24 hours. Determination of dry weight was carried out so that the final solution contained 30mg/L dry weight activated sludge.

Duration of test (contact time):

56 d

Initial conc.:

100 other: ppm

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was made up according to the OECD guideline and contained the following nutrients per litre of MilliQ: 85 mg of KH2PO4, 217.5 mg of K2HPO4, 334 mg of Na2HPO4.2H2O, 5 mg of NH4Cl, 22.5 mg of MgSO4.7H2O, 36.4 mg of CaCl2.2H2O, 0.25 mg of FeCl3.6H2O.
- Test temperature: 22 ºC
- Suspended solids concentration: All test vessels contained the prepared activated (30 mg/l) sludge inoculum in mineral medium.
- Continuous darkness: No

TEST SYSTEM
- Culturing apparatus: 250 mL serum bottles
- The bottles have an actual volume of 310 mL, and 100 mL of test solution was added to each bottle. The bottles were incubated at 22 ºC for up to 41 days with manometric respirometry data acquisition although the study was conducted for a total of 56 days with samples taken for analytical purposes only on days 41 and 56. The samples were stirred for the duration of the study with a magnetic stirrer.
- Number of culture flasks/concentration: Nine replicates of each set of vessels were prepared to allow for triplicate vessels to be sacrificed on two analytical sample occasions (days 41 and 56).
- Measuring equipment: WTW oxitopC (Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany) automatically calculates the consumption of oxygen.
- The test was carried out in nominal 250 mL serum bottles, sealed by a sensor head/CO2 trap in a temperature controlled incubator. As biodegradation progressed, the micro-organisms converted oxygen to carbon dioxide which was absorbed into the NaOH samples inside the flasks causing a net reduction in gas pressure within the respirometers.

SAMPLING
- Sampling frequency: 28, 41 and 56 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum blanks contained no test substance, in order to measure the endogenous respiration of the sludge, to demonstrate there was no other carbon source in the medium and to correct the relevant test vessels for baseline respiration. Additional blanks were prepared for extraction purposes.
- Abiotic sterile control: None
- A control was included (instead of a reference test substance) to directly observe the degradation of the parent substance test samples and to provide a direct comparison of degradation rate of this study against previous studies on the parent compound. As several tests had been conducted on the parent substance previously, only minor variations of degradation level attained between tests attests to the relative viability of the inoculum.

Reference substance:

not specified

Preliminary study:

Not applicable

Key result

Parameter:

% degradation (O2 consumption)

Value:

13.5

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

16.7

Sampling time:

41 d

Details on results:

Manometric Respirometry: Percentage biodegradation values for the test material at each time point (28 and 41 days) are provided in Table 5.2.1/2.
The parent substance attained a mean mineralisation value of 27% after 41 days (table 5.2.1/2), thereby confirming the inoculum viability and the non-Persistence of the test substance found in study report DBO R LSB 063.

Key result

Parameter:

ThOD

Value:

3.04 other: mg O2/mg substance

Table 5.2.1/1: Experimental design

 

Tests	Name	Planned mg		Real mg		Innoculated Mineral medium [L]
		Product	Ref	Product	Ref
1	Blank 1	-	-	-	-	0.1
2	Blank 2	-	-	-	-	0.1
3	Blank 3	-	-	-	-	0.1
4	Blank 1 / 28	-	-	-	-	0.1
5	TST 1/28	10	-	8.8	-	0.1
6	TST 2/28	10	-	10.4	-	0.1
7	TST 3/28	10	-	10.3	-	0.1
20	TST BUT 1/28	10	-	11.1	-	0.1
21	TST BUT 2/28	10	-	11.3	-	0.1
22	TST BUT 3/28	10	-	9.9	-	0.1
23	Blank 1 / 56	10	-	-	-	0.1
24	TST 1/56	10	-	10.4	-	0.1
25	TST 2/56	10	-	10.9	-	0.1
26	TST 3/56	10	-	9.2	-	0.1
39	TST BUT 1/56	10	-	9.3	-	0.1
40	TST BUT 2/56	10	-	10.7	-	0.1
41	TST BUT 3/56	10	-	10.9	-	0.1
Tests	Name	Planned ppm (pure product)		Real ppm (pure product)		Innoculated Mineral medium [L]
1	Blank 1	-	-	-	-	0.1
2	Blank 2	-	-	-	-	0.1
3	Blank 3	-	-	-	-	0.1
4	Blank 1 / 28	-	-	-	-	0.1
5	TST 1/28	100	-	88	-	0.1
6	TST 2/28	100	-	104	-	0.1
7	TST 3/28	100	-	103	-	0.1
20	TST BUT 1/28	100	-	111	-	0.1
21	TST BUT 2/28	100	-	113	-	0.1
22	TST BUT 3/28	100	-	99	-	0.1
23	Blank 1 / 56	100	-	-	-	0.1
24	TST 1/56	100	-	104	-	0.1
25	TST 2/56	100	-	109	-	0.1
26	TST 3/56	100	-	92	-	0.1
39	TST BUT 1/56	100	-	93	-	0.1
40	TST BUT 2/56	100	-	107	-	0.1
41	TST BUT 3/56	100	-	109	-	0.1

BLK: blank test

TST: Parent substance test

TST BUT: DD mercapto butyric acid test

For a molecule C_cH_hCl_clN_nNa_naO_oP_pS_swith a molecular weight MW, the ThOD is:

DThONH₃= (16[2c+1/2(h-cl-3n)+3s+5/2p+1/2na-o]mg/mg) / (MW)

For the parent substance, C₂₅H₄₆O₁S₁yields a molecular weight of 394.70 g/mol

For DD mercapto butyric acid, C17H28O3S yields a molecular weight of 312.47 g/mol

DThONH₃= 3.040 mgO2/mg substance.

 

Table 5.2.1/2: Biodegradation values for test compound and control Parent substance

Parent substance
Test	28 days	41 days	56 days	Sample analysis
TST 1/28	28	32	-	-
TST 2/28	20	24	-	-
TST 3/28	7	20	-	-
TST 1/56	23	29	-	-
TST 2/56	13	27	-	-
TST 3/56	26	30	-	-
DD mercapto butyric acid
Test	28 days	41 days	56 days	Sample analysis
TST BUT 1/28	15	17*	-	Day 41
TST BUT 2/28	23	26*	-	Day 41
TST BUT 3/28	1	1*	-	Day 41
TST BUT 1/56	15	18	-*	Day 56
TST BUT 2/56	14	19	-*	Day 56
TST BUT 3/56	13	19	-*	Day 56

 *: sacrificed for analysis of metabolites

-: No longer available (sacrificed)

 

Analytical Results

Only traces of remaining parent were found. In addition the following metabolites were detected: 2-mercapto acetic acid (A), Delta Damascone (B), DD mercapto acetic acid (C), DD mercapto propionic acid (D) and DD mercapto butyric acid (E)

 

All metabolites were characterized by comparison (GC retention time; MS) with authentic material.

 

Table 5.2.1/3: Metabolite concentration at day 41 and 56

 

Test ref.	Days	A [%]	B [%]	C [%]	D [%]	E [%]
RNE1574 / TST BUT 1.28 DBO R LSB 059 T20	41	0.56	5.16	66.79	0.00	0.00
RNE1569 / TST BUT 2.28 DBO R LSB 059 T21	41	0.26	3.71	80.87	0.19	0.59
RNE1566 / TST BUT 3.28 DBO R LSB 059 T22	41	0.24	3.21	90.37	0.16	1.16
average	41	0.35	4.03	79.34	0.12	0.58
RNE1592 / TST BUT 1.56 DBO R LSB 059 T39	56	0.49	7.34	62.65	0.18	0.00
RNE1595 / TST BUT 2.56 DBO R LSB 059 T40	56	0.43	4.06	56.89	0.00	0.00
RNE1598 / TST BUT 3.56 DBO R LSB 059 T41	56	0.39	3.47	61.48	0.00	0.32
average	56	0.44	4.96	60.34	0.06	0.11

The quantification is based on the GC peak area.

Validity criteria fulfilled:

no

Interpretation of results:

not readily biodegradable

Conclusions:

In an enhanced OECD 301 F biodegradation test the test material; DD mercapto butyric acid was found not to be persistent and underwent almost complete primary degradation during the study. Metabolite analysis shows that the test compound had already degraded to trace levels by the first sampling point at 41 days and was largely converted into a more polar and water soluble lower homologue, DD mercapto acetic acid. This degradation pathway, involving beta oxidation, is by far the most dominant one. A further substance, Delta Damascone, was also detected. The presence of DD at that end of the test could, in part, come from the partial decomposition of the parent substance (the registered substance) during the GC injection. But partial biodegradation of the registered substance leading to DD could not be fully excluded.
The principal metabolite - DD mercapto acetic acid - is slowly degraded further along non-specific pathways.

Executive summary:

The enhanced biodegradation test was performed using the Manometry Respirometry Method for ready biodegradability based on OECD 301 F Guideline with analytical follow-up (metabolites). This study was carried out as a supplementary test to a standard OECD 301F (DBO R Lsb063) in order to allow for analysis of a previously isolated reference substance metabolite at different incubation times. The study of this metabolite takes place in the context of the registered substance metabolic pathway investigation.

 

The test substance (Delta Damascone mercapto butyric acid (DD mercapto butyric acid)) was exposed to activated sludge (30 mg/L) at a concentration of 100 ppm with culture medium in closed bottles at 22 °C for 28/41/56 days. The degradation of the test material was assessed by the measurement of oxygen consumption.

 

The registered substance (the parent substance), i.e. DD mercapto dodecane was used as a control. No OECD recommended positive control was used.

 

Samples	After 28 days	After 41 days	After 56 days
Mean degradation *)	13.5%	16.7%	ND
Biodegradation product analysis	No	Yes	Yes

 

*)Six samples were used to provide the 28d and 41d manometric values (Test 20, 21, 22, 39, 40 & 41). Three samples were then sacrificed for analysis of potential biodegradation products hereafter referred to as metabolites after 28 days (Test 20, 21 & 22) and three others after 56 days (Test 39, 40 & 41)

 

Based on the results, test substance was found not to be persistent and underwent almost complete primary degradation during the study. Metabolite analysis shows that the test compound had already degraded to trace levels by the first sampling point at 41 days and was largely converted into a more polar and water soluble lower homologue, DD mercapto acetic acid. This degradation pathway, involving beta oxidation, is by far the most dominant one. A further substance, Delta Damascone, was also detected. The presence of DD at that end of the test could, in part, come from the partial decomposition of the registered substance during the GC injection. But partial biodegradation of registered substance leading to DD could not be fully excluded.

The principal metabolite - DD mercapto acetic acid - is slowly degraded further along non-specific pathways.

Description of key information

Weight of evidence approach:

Based on the experimental studies (performed according to OECD Guidelines), the registered substance is not considered readily biodegradable but a strong and consistent evidence of mineralisation to the main degradation product, Delta-Damascone mercapto acetic acid (DD-ma) was provided. All larger metabolites found (e.g. Delta-Damascone mercapto propionic acid (DD-mp)) can be considered as transient intermediates. Therefore, the registered substance is inherently biodegradable (not fulfilling specific criteria) and not persistent. The principal metabolite, DD-ma, is slowly degraded and in the absence of other biodegradation study on this metabolite, DD-ma is considered as potentially persistent (P) or very persistent (vP).

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

To assess the biotic degradation of the registered substance, a weight of evidence approach was performed with six ready biodegradability tests using different test methods.

The first study (Safepharm, 2004) was performed according to the OECD Guideline 301 B. The registered substance was not considered readily biodegradable. However, 26% of theoretical CO₂ production was reached after 28 days, indicating total primary biodegradation of the substance.

The second test (MSI, 2007) was performed according to the Japanese MITI National Standard, equivalent to the OECD Guideline 301 C. No biodegradation was measured. The pre-treatment steps of the inoculum, impacting the diversity of the inoculum, is a factor that can seriously impact the biodegradation and explain this result.

The third study (AkzoNobel, 2012) was performed according to a prolonged version of the OECD Guideline 301D. This test had been modified to permit prolonged measurements, and determination of biodegradation to both unadapted and adapted inocula. The assay was inoculated with river water, unacclimated activated sludge (from a wastewater treatment plant, predominantly domestic sewage), sludge from the SCAS unit and microorganisms from an enrichment culture. Approximately 20% of the substance carbon was found to be mineralised in the prolonged Closed Bottle test using unadapted activated sludge, unacclimated river water, and acclimated sludge from the SCAS unit, which supports the result of the OECD Guideline 301 B study. Acclimatization resulted in a decrease of the lag period but not an increase in percentage degradation. An enrichment culture was developed from activated sludge of the SCAS unit to ease analysis of the organic substances not susceptible to microbial degradation biodegradation. Microorganisms of the enrichment culture used as inocula in this test also degrade approximately 20% of the substance.

Then, a series of Manometric Respirometry tests following the recommendations of the OECD Guideline 301F were performed (Firmenich, 2012 and 2017) to assess the ultimate biodegradability of the registered substance and identifiy its metabolites. This assessment is present below and is attached as an official document in this endpoint summary.

The first OECD 301F study (Firmenich, 2012 - 28 days) was performed on the registered substance under standard test conditions over a period of 28 days. In this study, the test substance attained 16% (mean) degradation after 28 days. Therefore, the test substance cannot be considered to be readily biodegradable. Nevertheless, sufficient mineralisation occurred that complete primary degradation of the parent structure is ensured within this time frame.

A further OECD 301F study (Firmenich, 2012 - 84 days) was performed but with modifications of the standard guideline to determine and quantify the degradation products of the registered substance in order to identify and propose a degradation pathway. This method allowed a preliminary mass balance analysis over the study period which in this case was prolonged up to 84 days (with intermediate analysis points at 28 and 56 days). Based on the results, the test substance is not considered readily biodegradable but the study indicates total primary biodegradation of the substance and partial mineralisation. Therefore the substance can be considered as inherently biodegradable, not fulfilled specific criteria and not persistent. The main biodegradation products have been clearly identified by independent synthesis. The two main degradation products were Delta-Damascone mercapto acetic acid (DD-ma) (increasing over the study from 0.1% at day 28, 9.3% at day 56 and 25.6% at day 84) and Delta-Damascone mercapto proprionic acid (DD-mp) (increasing over the study from 5.4% at day 28, 8.7% at day 56 and 14.4% at day 84). Delta-Damascone (DD) was found at varying percentages between 3 and 11% with no consistent trend.

In a third OECD 301F study (Firmenich, 2017), one of the degradation products of the registered substance, Delta-Damascone mercapto butyric acid (DD-mb), was isolated and tested using the OECD Guideline with modifications to the standard method again in order to analyse and quantify major metabolic products over a period of 56 days with intermediate analyses at 28 and 41 days. The reason for the choice of this degradation product (rather than the more prevalent metabolites, DD-ma or DD-mp, found in the previous study) was that, prior to taking the decision to perform a full metabolite study in May to August 2012, an in silico tool, the EAWAG biocatalysis/biodegradation database was used to predict probable degradation products of the parent and this structure was considered to be one of the major metabolites. A study was therefore performed on DD-mb before undertaking the 84-day study. According to the results of this test, DD-mb was found not to be persistent and underwent almost complete primary degradation during the study. Metabolite analysis shows that the test compound had already degraded to trace levels by the first sampling point at 41 days and was largely converted into a more polar and water soluble lower homologue, DD-ma. This degradation pathway, involving beta oxidation, is by far the most dominant one. A further substance, Delta-Damascone (DD), was also detected. The presence of DD at that end of the test could, in part, come from the partial decomposition of the parent substance (the registered substance) during the GC injection. But partial biodegradation of the registered substance leading to DD could not be fully excluded. The principal metabolite, DD-ma, is slowly degraded further along non-specific pathways.

In conclusion, based on these studies, the registered substance is not considered readily biodegradable but a strong and consistent evidence of mineralisation to the main degradation product, DD-ma, was provided. Therefore, the registered substance is inherently biodegradable (not fulfilling specific criteria) and not persistent. All larger metabolites found in the studies (e.g. DD-mp) can be considered as transient intermediates. The degradation pathway can therefore be considered as a reduction of the alkyl chain by beta oxidation resulting in a DD mercapto short chain carboxylic acid. As for other carboxylic acids, DD-ma readily dissociates into a carboxylate anion and a positively charged hydrogen ion (proton). The dissociation constant of DD mercapto alcanoic acids can be estimated to be between 3 and 4. This estimation is based on a read-across with 2 -mercapto acetic acid, having at 25°C a dissociation constant for pK1 of 3.6 (source: Kirk-Othmer Encyclopedia of Chemical Technology. John Wiley & Sons, Inc). Even in the undissociated form, DD-ma is a quite polar compound (log Kow = 3.1 [arithmetical mean value of QSAR results]) and therefore, even if further degradation of this substance has not been demonstrated in these experiments, the final degradation product analysed in this study, DD-ma, is not expected to bioaccumulate significantly in the environment. It is believed that the concentrations of DD measured in the residue studies (Firmenich, 2012) occurred as a result of extensive thermal degradation in the injection of the metabolites and remaining test compound. This theory is consistent with the absence of any concentration trend over time in both metabolite studies. DD is therefore not considered to be a likely candidate degradation products for the registered substance.