Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20th July 2010 to 18 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15th September 2009 Date of signature: 26th November 2009

Test material

Test material information

Constituent 1

Reference substance name:

[4-(2-methylbutan-2-yl)phenyl]x[2,4-bis(2-methylbutan-2-yl]3-x phosphite (where x = 0, 1, 2, 3)

IUPAC Name:

[4-(2-methylbutan-2-yl)phenyl]x[2,4-bis(2-methylbutan-2-yl]3-x phosphite (where x = 0, 1, 2, 3)

Details on test material:

Sponsor's identification :DVS005u (aka Weston 705)
Description : clear colourless viscous liquid
Batch number : MW9F23A901
Date received : 5 November 2009
Expiry date : 15 January 2011
Storage conditions :approximately 4ºC in the dark
Carbon Analysis :77.11% wt/wt** (mean of 2 replicate values)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

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 19 July 2010 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: not stored, as used on day of collection

- Storage length: not applicable

- 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 21ºC 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 filter three successive times with 10 ml of deionised reverse osmosis water. 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 concentration was equal to 2.9 g/l prior to use.

- Pretreatment: as above

- Concentration of sludge: not stated

- Initial cell/biomass concentration: 13.0 mg/l, equivalent to 10 mg carbon/l

- Water filtered: yes

- Type and size of filter used, if any: pre-weighed GD/A filter paper

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 other: mg carbon/l

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
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 MgSO4.7H2O 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: Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

- Solubilising agent (type and concentration if used): none used
- Test temperature: 21°C
- pH: 7.5
- pH adjusted: no
- CEC (meq/100 g): not stated in report
- Aeration of dilution water: not applicable
- Suspended solids concentration: 2.9 mg/l prior to use. Each test vessel was inoculated with the prepared inoculum to give a final concentration of 30 mg suspended solid (ss)/l.
- Continuous darkness: yes


TEST SYSTEM
Culturing apparatus:
- Number of culture flasks/concentration: Two (duplicate)
Preparation of test system
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 reference item (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 item, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
d) The test item plus the reference item in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Silica gel was added to the control and reference item vessels in order to maintain consistency between these vessels and the test item vessels.
Each test 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°C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 ml of culture medium and 31.0 ml of inoculum and aerated overnight. On Day 0 the test and reference items 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 self-indicating 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.

- 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 continuously 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. The CO2 absorbing solutions were prepared using purified de-gassed water.

- 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: he 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:
CO2 analysis
Samples (2 ml) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.

Dissolved organic carbon (DOC) analysis
Samples (20 ml) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item 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.

- Sampling method:
CO2 analysis:
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. Samples (300 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
On Days 0 and 28 samples (20 ml) were removed from the control and reference item vessels and 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°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 reference 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 described in report
- Sample storage before analysis: Not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: None
- Abiotic sterile control: None
- Toxicity control: For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (39.0 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No.540085-438) prior to dispersal in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm,
15 minutes). The test item/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 3 litres to give a final concentration of 13.0 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

STATISTICAL METHODS: None

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

Preliminary study:

Not applicable

Test performance:

The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

20

Sampling time:

28 d

Remarks on result:

other: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Details on results:

The test item attained 20% 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 33% degradation after 14 days and thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 71% degradation after 14 days 87% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 100% and 98% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 71% degradation after 14 days 87% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions

Any other information on results incl. tables

Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg IC)				Sodium Benzoate (mg IC)				Test Item (mg IC)				Test Item plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.28	1.28	1.40	1.17	1.40	1.28	1.17	1.05	1.17	1.17	1.40	1.40	1.17	1.28
2	5.22	-	5.22	-	19.37	-	18.33	-	6.38	-	5.22	-	15.66	-
6	9.69	-	11.30	-	30.91	-	31.83	-	11.19	-	9.92	-	28.95	-
8	13.76	-	14.68	-	37.04	-	36.12	-	13.76	-	14.10	-	36.23	-
10	10.94	-	15.50	-	37.62	-	36.82	-	14.48	-	14.71	-	36.94	-
14	16.77	-	18.93	-	38.87	-	39.67	-	15.98	-	17.45	-	37.85	-
21	17.91	-	17.80	-	45.74	-	44.95	-	19.94	-	24.56	-	-	-
28	24.53	-	22.96	-	44.91	-	46.14	-	27.55	-	30.91	-	-	-
29	26.61	0.93	26.83	1.04	52.33	0.81	53.22	1.04	30.50	0.81	35.18	0.81	-	-

 

 Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
2	45	2	17
6	70	0	31
8	75	0	37
10	80	5	40
14	71	0	33
21	92	15	-
28	73	18	-
29*	87	20	-

 

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

Test vessel	Total Carbon* (mg/l)	Inorganic Carbon* (mg/l)	IC Content (% of TC)
Sodium Benzoate 10 mg C/lR1	10.46	0.43	4
Sodium Benzoate 10 mg C/l R2***	8.58	0.11	1
Test Item 10 mg C/l R1	10.57**	0.41	4
Test Item 10 mg C/l R2	10.24**	0.30	3
Test Item plus Sodium Benzoate Toxicity Control 20 mg C/l	20.39**	0.21	1

 

R₁– R₂= Replicates 1 and 2

Abs= CO₂absorber vessel

- = No value determined

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

- = No degradation result obtained due to toxicity control being terminated after 14 days.

R₁– R₂= Replicates 1 and 2

*Corrected for control values.

**Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable

***Duplicate sample analysed as the original sample result was deemed anomalous

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable

Conclusions:

The test material attained 20% 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 item in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO₂Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

Methods. The test item, at a concentration of 10 mg Carbon/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 (Handleyet al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

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

Results. The test item attained 20% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.