Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

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:

Between 25 October 2011 and 24 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP; appropriate adaptations to ensure bioavailability made according to guideline and generally accepted recommendations

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Remarks:

- Following ISO 10634 (1995) and literature recommendations the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper, which was broken up by high shear mixing.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Remarks:

- see EU C.4 entry

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998, Paragraph (M)

Deviations:

no

Remarks:

- see EU C.4 entry

Qualifier:

according to guideline

Guideline:

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

Deviations:

not applicable

Remarks:

Appropriate elements considered

Principles of method if other than guideline:

Following the recommendations of ISO 10634 (1995) and the pre-study solubility work conducted, the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper. High shear mixing was also applied to break up the filter paper containing the test item (Handley et al 2002). This method was used in order to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to test organisms.
- Handley JW, Mead C, Rausina GA, Waid LJ, Gee JC, Herron SJ (2002). The Use of Inert Carriers in Regulatory Biodegradation Tests of Low Density Poorly Water-soluble Substances. PMID 12146631 Chemosphere 48:529-34.

GLP compliance:

yes

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained on 24 October 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, U.K., which treats predominantly domestic sewage.
- Storage conditions: The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC
- Storage length: < 1 d
- 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.
- Concentration of sludge: The suspended solids concentration was equal to 4.1 g/L prior to use.
- Water filtered: No

Duration of test (contact time):

28 d

Initial conc.:

11.4 mg/L

Based on:

ThIC

Initial conc.:

15.5 mg/L

Based on:

test mat.

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 Guidelines.
Solution a: 8.50 g KH2PO4/L, 21.75 g K2HPO4/L, 33.40 g Na2HPO4.2H2O/L, 0.50 g NH4Cl/L; pH = 7.4
Solution b: 27.50 g CaCl2/L
Solution c: 22.50 g MgSO4.7H2O/L
Solution d: 0.25 g FeCl3.6H2O/L
10 mL of Solution a, 1 mL of Solution b, 1 mL of Solution c and 1 mL of Solution d were added to purified water (reverse osmosis purified and deionised water, Elga Optima 15+ or Elga Purelab Option R-15 BP) to reach a 1 L final volume.
- Additional substrate: No
- Solubilising agent: Initially the test item was dissolved in acetone. This solvent stock solution was dispensed onto a filter paper and the solvent was allowed to evaporate to dryness for approximately 15 min, before high shear mixing and addition to the test vessel. Thus no solubilising agent was present in the test medium.
- Test temperature: 21 °C
- pH: At day 28 in the two replicates of the treatments and the inoculum blanks pH 7.5 and in the procedure controls pH 7.6 was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
- pH adjusted: No
- Aeration of dilution water: Approximately 24 h prior to addition of the test and reference substances the vessels were filled with 2400 mL of culture medium and 22.0 mL of inoculum and aerated overnight. The remaining medium added to reach 3 L volume was not aerated.
- Suspended solids concentration: 30 mg suspended solids/L (final concentration in test vessels)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels each containing 3 L of solution
- Experimental Preparation: The test item was prepared as a solvent stock solution and an aliquot dispensed onto filter paper prior to high shear mixing and dispersion in culture 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. An amount of test item (1000 mg) was dissolved in 10 mL of acetone to give a 1000 mg/10 mL solvent stock solution. An aliquot (465 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 min. The filter paper was dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 min) prior to addition to inoculated culture medium. The volume was then adjusted to 3 L to give a final concentration of 15.5 mg/L, equivalent to 11.4 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution. A filter paper with acetone evaporated to dryness was added to the control and reference item vessels in order to maintain consistency between these vessels and the test item vessels.

- Number of culture flasks/concentration:
a) A control, in duplicate, consisting of inoculated culture medium plus a GF/A filter paper (70 mm diameter) with acetone evaporated off
b) The reference substance (sodium benzoate), in duplicate, in inoculated culture medium plus a GF/A filter paper (70 mm diameter) with acetone evaporated off to give a final concentration of 10 mg carbon/L
c) The test item on a GF/A filter paper (70 mm diameter) with acetone evaporated off, in duplicate, in inoculated culture medium to give a final concentration of 11.4 mg carbon/L.
d) The test item on a GF/A filter paper (70 mm diameter) with acetone evaporated off plus the reference substance in inoculated culture medium to give a final concentration of 21.4 mg carbon/L to act as a toxicity control (one vessel only)
- Method used to create aerobic conditions: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer. The flow rates were adjusted to 40 mL/min where necessary. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (carbosorba) granules.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- Details of trap for CO2 (no organic volatile traps were 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: To perform the CO2 analysis the first CO2 absorber vessels from the control, reference and test item were sampled on days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and the toxicity control first CO2 absorber vessel were sampled 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.
Test media samples were taken on day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content. 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 (30 mL) were removed from the control and reference item vessels prior to DOC analysis.
- Sampling method: For CO2 analysis 2 mL samples were taken from the CO2 absorber vessels. 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. For DOC analysis samples (30 mL) were removed from the test item and toxicity control vessels.
- Sample storage before analysis: No, the samples were analysed for CO2 immediately.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates (only inoculum, no added carbon source and filter paper); control vessels were prepared containing a single filter paper (GF/A, 70 mm diameter) per 3 L of inoculated culture medium. Acetone (465 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 min prior to the addition to inoculated culture medium in order to maintain consistency between the control and test item vessels.
- Abiotic sterile control: No
- Toxicity control: 1 replicate (inoculum, reference substance sodium benzoate, test item and filter paper); 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 aliquot (465 µL) of the test item solvent stock solution was dispensed onto a filter paper GF/A, 70 mm diameter with acetone evaporated off and the solvent allowed to evaporate for approximately 15 min. The filter paper was dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 min) prior to addition to the test vessel containing inoculated culture medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 L to give a final concentration of 15.5 mg test item/L plus 17.13 mg sodium benzoate/L equivalent to a total of 21.43 mg carbon/L.
- Other: 2 replicates procedure control (inoculum, reference substance sodium benzoate and filter paper); for the purposes of the test, a reference substance, sodium benzoate, was used. An initial stock solution of 1000 mg/L was prepared by dissolving the reference substance directly in culture medium with the aid of ultrasonication for approximately 5 min. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated culture medium and the volume adjusted to 3 L to give a final test concentration of 17.13 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference substance was inverted several times to ensure homogeneity of the solution. A filter paper (GF/A 70 mm diameter) was added to each vessel in order to maintain consistency between the test and reference substance vessels. Acetone (465 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 min. The filter paper was dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 min) prior to addition of each vessel.

Reference substance:

benzoic acid, sodium salt

Remarks:

(C6H5COONa), Sigma Aldrich Lot No MKBC1469 in a concentration of 17.13 mg/L corresponding to 10 mg ThIC/L

Preliminary study:

As the test item is water insoluble pre-study solubility/dispersibility work was performed in order to determine the most suitable method of preparation (see overall remarks section).

Test performance:

The total CO2 evolution in the control vessels on day 28 was 18.94 mg/L and therefore satisfied the validation criterion given in the guidelines, as 40 mg/L medium were not exceeded (validity criterion 1).
The IC content of the test item culture medium at the start of the test (see Table 4 below) was below 5 % of the TC content and hence satisfied the validation criterion given in the guidelines (validity criterion 2).
The difference between the values for CO2 production at the end of the test for the replicate vessels was < 20 % and hence satisfied the validation criterion given in the guidelines (validity criterion 3).

Parameter:

% degradation (CO2 evolution)

Value:

68

Sampling time:

28 d

Details on results:

The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29, i.e. after acidification, showed a decrease in all replicate vessels with the exception of test item replicate R2. This decrease was considered to be due to sampling/analytical variation.
Inorganic carbon analysis of the samples from the second absorber vessels on day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred (Table 2).
The test item attained 68 % degradation after 28 d and obviously the degradation process was still ongoing as no plateau formation was visible (see illustration below). The test item did not satisfy the 10-day window validation criterion, whereby 60 % degradation must be attained within 10 d of the degradation exceeding 10 %. However, the test item has exhibited potential for rapid and ultimate degradation and the applicability of the 10-day window for UVCB need to be negated with reference to further guidance documents (see discussion in executive summary).
The toxicity control attained 55 % degradation after 14 d thereby confirming that the test item was not toxic to the sewage treatment microorganisms used in the test (validity criterion 4).

Results with reference substance:

In the procedure control Sodium benzoate attained 65 % degradation after 14 d and 93 % degradation after 28 d thereby confirming the suitability of the inoculum and test conditions (validity criterion 5).
Analysis of the test media taken from the reference item culture vessels on days 0 and 28 for Dissolved Organic Carbon (DOC), (see Table 5 below), gave percentage degradation values of 100 % for replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were slightly 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.

Table 2: Inorganic Carbon (IC) Values on Each Analysis Occasion

Day	Control, inoculum blank [mg IC]				Sodium Benzoate, Procedure control [mg IC]				Test Item [mg IC]				Test Item plus Sodium Benzoate, Toxicity Control [mg IC]
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2
0	0.70	0.58	0.70	0.70	0.70	0.70	0.47	0.47	0.70	0.70	0.70	0.58	0.58	0.70
2	5.22	-	3.83	-	17.98	-	16.70	-	3.94	-	4.52	-	12.88	-
6	10.27	-	8.65	-	26.18	-	26.53	-	10.03	-	13.84	-	20.41	-
8	13.42	-	10.89	-	30.85	-	30.27	-	17.77	-	21.56	-	23.85	-
10	14.48	-	10.60	-	29.52	-	29.52	-	19.84	-	20.52	-	43.55	-
14	14.96	-	12.92	-	33.66	-	32.98	-	26.29	-	26.41	-	49.53	-
21	15.55	-	14.87	-	37.52	-	36.73	-	29.52	-	29.97	-	-	-
28	16.57	-	14.45	-	45.13	-	45.81	-	37.30	-	39.09	-	-	-
29	15.70	2.44	14.03*	2.78	41.31	2.78	43.98	2.78	37.07	2.44	39.19	2.67	-	-

R1, R2 = Replicates 1 and 2

Abs = CO2 first and second absorber vessels (Abs1 and Abs2, respectively)

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

Table 3: Percentage Biodegradation Values

Day	% Degradation
	Sodium Benzoate, Procedure Control	Test Item	Test Item plus Sodium Benzoate, Toxicity Control
0	0	0	0
2	43	0	13
6	56	7	17
8	61	22	18
10	57	22	48
14	65	36	55
21	73	43	-
28	100	66	-
29*	93	68	-

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

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Table 4: 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/L R1	9.52	-0.01	0
Sodium Benzoate 10 mg C/L R2	9.39	-0.01	0
Test Item 11.4 mg C/L R1	11.03**	-0.03	0
Test Item 11.4 mg C/L R2	11.08**	-0.02	0
Test Item plus Sodium Benzoate Toxicity Control 21.4 mg C/L	20.95**	-0.03	0

R1, R2 = Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentrations being less than 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

Table 5: 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	9.53	95	< control	0	100
Sodium Benzoate 10 mg C/L R2	9.40	94	< control	0	100

R1, R2= Replicates 1 and 2

* Corrected for control values

Observations on the Test Preparations Throughout the Test Period

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout were observed in all vessels. No undissolved test or reference item was visible at any observation time (days 0, 6, 13, 20 and 27).

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

10-day window criterion not applicable due to UVCB character; rapid, ready and ultimate aerobic biodegradation in water

Executive summary:

The biodegradation of the test item in water was determined in a GLP-compliant study by screening using the carbon dioxide evolution method according to the EU C.4-C (2008), OECD TG 301 B (1995) and OPPTS 835.3110 (1998) standards. Appropriate adaptations to ensure bioavailability were made according to the ISO 10634 (1995) guideline and generally accepted literature recommendations. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

The test item, at a concentration of 15.5 mg/L corresponding to 11.4 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 d. Following the recommendations of the ISO 10634, the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item was evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby enabling the potential for biodegradation. A filter paper with acetone evaporated to dryness was added to the control and reference item vessels in order to maintain consistency between these vessels and the test item vessels. The degradation of the test item was assessed by the determination of carbon dioxide produced. The control and blank system comprised inoculum blank to define the background carbon dioxide production, the procedure control to demonstrate the suitability of the micro-organisms to degrade the reference substance, sodium benzoate and the toxicity control to enable the detection of toxicity to micro-organisms. Each control was performed in two replicates, except the toxicity control.

The controls confirmed the appropriateness of the test system and the absence of sewage sludge micro-organism toxicity at 15.5 mg/L. The test item attained 68 % degradation after 28 d and no plateau formation was visible yet, indicating further ongoing degradation. Under the strict terms and conditions of OECD TG 301 B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-day window validation criterion, whereby 60 % degradation (as a respirometric parameter was followed) must be attained within 10 d of the degradation exceeding 10 %. However, the test item has exhibited the potential for rapid and ultimate degradation. Considering the fact that the test guidance criteria refer to pure substances and the probably varying lag times of the individual constituents the UVCB test item, it is anticipated that a sequential biodegradation of the individual structures is taking place. According to OECD (2006) the 10-day window should not be applied to interpret the results of the test in such cases. Accordingly the UVCB test item can be regarded as rapidly, readily and ultimately biodegradable.

• OECD Organisation for Economic Co-operation and Development (2006). Revised Introduction to the OECD Guidelines for Testing of Chemicals. Section 3. Part 1: Principles and Strategies Related to the Testing of Degradation of Organic Chemicals. Self-published. Adopted 23 March 2006. 17 p.

Description of key information

Readily biodegradable (10-day window criterion not applicable due to UVCB character; rapid, ready and ultimate aerobic biodegradation in water) (OECD 301 B)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The biodegradation in water was determined in a GLP-compliant study (Clarke 2011, Harlan Report no. 41103271) by screening using the carbon dioxide evolution method according to the EU C.4-C (2008), OECD TG 301 B (1995) and OPPTS 835.3110 (1998) standards. Appropriate adaptations to ensure bioavailability were made according to the ISO 10634 (1995) guideline and generally accepted literature recommendations. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

Biodegradation was followed by measurement of the respirometric parameter carbon dioxide evolution under aerobic conditions. The pass level for this test is 60 % ThCO2, which was clearly met as 68 % biodegradation was observed in 28 d. According to the following discussion no 10-day window applies as the submission item is a UVCB mixture of structurally similar chemicals.

With reference to CLP, the text of the regulation EC (2011, p L 83/24) is as follows: “These levels of biodegradation must be achieved within 10 days of the start of degradation which point is taken as the time when 10 % of the substance has been degraded, unless the substance is identified as an UVCB or as a complex, multi-constituent substance with structurally similar constituents. In this case, and where there is sufficient justification, the 10-day window condition may be waived and the pass level applied at 28 days.”

The assessment has to be based on ECHA (2008, section R.7.9.4.1 Laboratory data on degradation/biodegradation, p 171) stating: “These pass levels have to be reached in a 10-day window within the 28-day period of the test. The 10-day window does not apply to TG 301 C or if the test substance represents a mixture of homologous compounds e.g. technical surfactants. The 10-day window begins when the degree of biodegradation has reached 10 % DOC removal, ThOD or ThCO2 and must end before or at day 28 of the test. The pass levels of either 60 % ThOD (or ThCO2) or 70 % DOC removal practically represent complete ultimate degradation of the test substance as the remaining fraction of 30-40 % of the test substance is assumed to be assimilated by the biomass or present as products of biosynthesis (OECD 2006).” The relevant paragraphs referred to are OECD (2006, paragraph 43, p 8): “Although these tests are intended for pure chemicals, it is sometimes relevant to examine the ready biodegradability of mixtures of structurally similar chemicals like oils and surface-active substances (surfactants). Such substances often occur as mixtures of constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms. Testing of each individual component may be costly and impractical. If a test on the mixture is performed and it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” and OECD (2006, paragraph 17, p 4): “The pass levels of either 60 % ThOD (or ThCO2) or 70 % DOC removal practically represent complete ultimate degradation of the test substance as the remaining fraction of 30-40 % of the test substance is assumed to be assimilated by the biomass or present as products of biosynthesis.”

Therefore it is to conclude that that 10-day window criterion drops and the submission item biodegradability is evidenced to show rapid, ready and ultimate biodegradation.

• EC European Commission (2011). Commission Regulation (EU) No 286/2011 of 10 March 2011 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures. Section 4.1.2.9.5. (a) Official Journal of the European Union 30.3.2011.
• ECHA European Chemicals Agency (2008). Guidance on information requirements and chemical safety assessment. Chapter R.7b: Endpoint specific guidance. Guidance for the implementation of REACH. Self-published May 2008. 228 p.
• OECD Organisation for Economic Co-operation and Development (2006). Revised Introduction to the OECD Guidelines for Testing of Chemicals. Section 3. Part 1: Principles and Strategies Related to the Testing of Degradation of Organic Chemicals. Self-published. Adopted 23 March 2006. 17 p.