Reaction mass of 3-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-2-hydroxypropyl 2methylprop-2-enoate and 1-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-3-hydroxypropan-2-yl 2-methylprop-2-enoate

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:

8 July 2008 to 3 September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to current OECD giudelines and to GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Test material adsorbed onto an inert support prior to dispersion in the test vessels

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels. Using this method the test material is evenly distributed throughout the test medium and the surface area of the test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 4th August 2008 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: ~21 deg C
- Storage length: Used on day of collection.
- Preparation of inoculum for exposure: The activated sewage sludge sample 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. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 deg C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105 deg C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/l prior to use.
- Pretreatment: as above
- Concentration of sludge: 3.0 g/k
- Initial cell/biomass concentration: as above
- Water filtered: yes
- Type and size of filter used, if any: Filtered by suction through GF/A filter paper using a Buchner funnel.

Duration of test (contact time):

28 d

Initial conc.:

5 other: mg C/l

Based on:

other: Initial experiment conducted at 10 mg C/l exihibited inhibitory effects. Therefore, following recomendations of the Test Guideline, in the definative test, the test material was at a reduced concentration of 5 mg C/l.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The culture medium used in this study was that recommended in the OECD guidelines.
Culture medium:
Solution a: KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l
pH: 7.4

Solution b: CaCl2 27.50 g/l
Solution c: MgSO4.7H20 22.50 g/l
Solution d: FeCl3.6H2O 0.25 g/l

To 1 litre (final volume) of purified water was added the following volumes of solutions a-d
10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of solution d

- Additional substrate: None
- Solubilising agent (type and concentration if used): None used
- Test temperature: 21 deg C
- pH: ~8
- pH adjusted: no
- CEC (meq/100 g): Not available
- Aeration of dilution water: N/A
- Suspended solids concentration: 30 mg/l
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 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 plus 100 mg silica gel.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 5 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 15 mg carbon/l to act a a toxicity control (one vessel only).
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels. Each teste vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21 deg 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 30 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.

- Method used to create aerobic conditions: The culture vessels were sealed and CO2 free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continously by magnetic stirrer.
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: 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.


SAMPLING
- Sampling frequency: Samples (2ml) were taken from the first CO2 absorber vessel on Days 0,1,2,3,6,8,10,12,14,16,18,22,24,27,28 and 29. The second absorber vessel was sampled on Days 0 and 29.
- Sampling method: The samples taken on Days 0,1,2,3,6,8,10,14,16,22,27,28 and 29 were analysed for CO2 immediately. The samples taken on Days 12, 18 and 24 were stored at approximately -20 deg C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed no significant change in degradation occured during this time and therefore additional analyses were considered unneccessary.

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.

The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-Vcsh TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthphosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

-Dissolved organic carbon (DOC analysis):
Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680 deg C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.


- Sterility check if applicable: Not available.
- Sample storage before analysis: Samples taken on Days 12, 18 and 24 were stored at approximately -20 deg C.
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel was prepared.
- Abiotic sterile control: Not available
- Toxicity control: The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 15 mg carbon/l to act a a toxicity control (one vessel only).
- Other: non


STATISTICAL METHODS:
Statistical analysis of the Day 29 Inorganic Carbon (IC) values for the control and test material vessels was carried out using a Student's t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999-2001).

Reference substance:

benzoic acid, sodium salt

Preliminary study:

In an initial experiment conducted at a concentration of 10 mg C/l, the toxicity control vessel, containing both the test material and sodium benzoate, attained less than 25% biodegradation after 14 days. These results indicated that the test material exhibited inhibitory effects at this concentration.

Test performance:

In the definative test, the test material concentration was reduced to 5 mg C/l to overcome any possible inhibitory effects. It was not possible to test at concentrations below 5 mg C/l as below this concentration it is not possible to distinguish between background CO2 evolution from the inoculum and CO2 evolution due to biodegration when using inorganic carbon analysis.

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

The test material attained 0% degradation afer 28 days and therefore cannot be considered readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The total CO2 evolution in the control vessels on Day 28 was 31.66 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Results with reference substance:

Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see table below, gave percentage degradation values of 100% for both Replicates R1 and R2.

Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions and the contents of the standard material vessels to be slightly cloudy light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were cloudy dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a cloudy dispersion with no undissolved standard material or test material visible.

Dissolved Organic Carbon (DOC) Values in the CUlture Vessels on Days 0 and 28

Test Vessel	DOC* Concentration
	Day 0		Day 28
	mg C/l	% of Nominal Carbon Content	mg C/l	% of Initial Carbon Concentration	% Degradation
Sodium Benzoate 10 mg C/l R1	9.95	100		0	100
Sodium Benzoate 10 mg C/l R2	10.14	101		0	100

R₁ – R₂ = Replicates 1 and 2

* Corrected for control values

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

See below table for percentage biodegradation values for test material and refernce substance.

                Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	21	10	13
2	40	4	9
3	43	0	10
6	54	16	9
8	52	4	11
10	73	0	9
14	82	4	9
16	82	0	7
22	92	0	0
27	87	0	0
28	94	0	0
29*	102	0	0

* Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 0% 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

In an initial experiment conducted at a concentration of 10 mg C/l, the toxicity control vessel, containing both the test material and sodium benzoate, attained less than 25% biodegradation after 14 days.  These results indicated that the test material exhibited inhibitory effects at this concentration.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test material at a reduced concentration of 5 mg C/l was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

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 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 9% degradation after 14 days and 0% degradation after 28 days thereby indicating that, even at the reduced concentration of 5 mg C/l, the test material was exhibiting inhibitory effects on the sewage treatment micro-organisms used in the test.  It was not practical to further reduce the test concentration employed in the study below 5 mg C/l as it would not be possible to distinguish between background CO₂ evolution from the inoculum and CO₂ evolution due to biodegradation using inorganic carbon analysis.

Care should be taken in the interpretation of the results due to the toxic nature of the test material to the activated sewage sludge micro-organisms used in the study.

Description of key information

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

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

Inan initial experiment conducted at a concentration of 10 mg C/l, the toxicity control vessel, containing both the test material and sodium benzoate, attained less than 25% biodegradation after 14 days.  These results indicated that the test material exhibited inhibitory effects at this concentration.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test material at a reduced concentration of 5 mg C/l was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

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 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 9% degradation after 14 days and 0% degradation after 28 days thereby indicating that, even at the reduced concentration of 5 mg C/l, the test material was exhibiting inhibitory effects on the sewage treatment micro-organisms used in the test.  It was not practical to further reduce the test concentration employed in the study below 5 mg C/l as it would not be possible to distinguish between background CO₂evolution from the inoculum and CO₂evolution due to biodegradation using inorganic carbon analysis.

Care should be taken in the interpretation of the results due to the toxic nature of the test material to the activated sewage sludge micro-organisms used in the study.