Phosphoric acid, mono- and di-C6-12-(even numbered)-alkyl esters, potassium salts

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From August 16, 2011 to September 15, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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 the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

Refer to section 13 for details on the read-across justification. The biodegradation study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Test Species: A mixed population of activated sewage sludge micro-organisms was obtained on 15 August 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of inoculum: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 3.4 g/l prior to use.* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven Culture mediumThe culture medium used in this study (see below) was that recommended in the OECD Guidelines.

Culture Medium:The culture medium used in this study was that recommended in the OECD Guidelines.

Solution a KH2PO48.50 g/lK2HPO421.75 g/lNa2HPO4.2H2O33.40 g/lNH4Cl0.50 g/lpH = 7.4
Solution b CaCl227.50 g/L
Solution c MgSO4.7H2O22.50 g/L
Solution d FeCl3.6H2O0.25 g/L To 1 L (final volume) of purified water* was added the following volumes of solutionsa – d.10 mL of Solution a1 mL of Solution b 1 mL of Solution c 1 mL of Solution d* Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

Duration of test (contact time):

28 d

Initial conc.:

10.3 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Pre-Study solubility work: Data supplied by the Sponsor indicated that the test substance was dispersible in water. Prestudy solubility work confirmed by Dissolved Organic Carbon analysis that it was not possible to attain a homogenous dispersion of the test substance by using ultrasonication.In order to obtain the best test able dispersion of the test substance, further pre-study solubilityl dispersibility work was performed (see details in any other information on materials and methods including tables section).

Experimental Preparation: For the purpose of the test, the test susbtance was dispersed directly in culture medium.An amount of test item (55.2 mg) was dispersed in approximately 400 ml of culture medium with the aid of ultrasonication for 15 minutes prior to dispersal in inoculated culture medium. The volume was adjusted to 3 litres to give a final concentration of 18.4 mg/L, equivalent to 10.3 mg carbon/L. A test concentration of 10.3 mg carbon/L was employed in the test following the recommendations of the test guideline.

Reference Item: For the purposes of the test, a reference item, sodium benzoate (C6H5COONa), was used. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in culture medium with the aid of ultrasonication for approximately 2 minutes. 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 litres to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity control: For the purposes of the test, a toxicity control, containing the test substance and sodium benzoate, was prepared in order to assess any toxic effect of the test substance on the sewage sludge micro-organisms used in the susbtance. An amount of test substance (55.2 mg) was dispersed in approximately 400 mL of culture medium with the aid of ultrasonication for 15 minutes prior to dispersal in inoculated culture medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 18.4 mg test substance/L plus 17.1 mg sodium benzoate/l, equivalent to a total of 20.3 mg carbon/L.

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.
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L.
c) The test substance, in duplicate, in inoculated culture medium to give a final concentration of 10.3 mg carbon/L.
d) The test substance plus the reference item in inoculated culture medium to give a final concentration of 20.3 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 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 substance the vessels were filled with 2400 ml of culture medium and 26.5 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.

Sampling and analysis: CO2 analysis: Samples (2 mL) taken from the control, reference and test substances first CO2 absorber vessels on Days 0, 3, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 3, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test substance and on Day 0 for the toxicity control. The samples taken on Days 0, 3, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately. 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. Samples (300 or 50 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. Each analysis was carried out in triplicate.Dissolved organic carbon (DOC) analysis:Samples (30 mL) were removed from the test substance and toxicity control vessels on Day 0 prior to the addition of the test substance in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (approximately 5 mL discarded) prior to DOC analysis. DOC analysis of the test substance dispersions after dosing was not possible due to the insoluble nature of the test substance in water. On Days 0 and 28 samples (30 mL) were removed from the control and reference substance vessels and filtered through 0.45 µm Gelman AcroCap filters (approximately 5 mL discarded) prior to DOC analysis.The samples were analysed for DOC using a Shimadzu TOC-VCSH TOC analyser and a Shimadzu TOC-VCPH TOC Analyser. Samples (50 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.

pH measurements: The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Evalaution of data: Please see attached evaluation of data (calculation of carbon content, percentage degradation) in background material section.

Validation criteria: The results of the degradation test are considered valid if in the same test the reference substance yields eqault to or greater than 60% degradation by Day 14. The test item may be considered to be readily biodegradable if greater than or equal to 60% degradation is attained within 28 d. This level of degradation must be reached within 10 d of biodegradation exceeding 10%. The toxicity control (test substance and sodium benzoate) should attain equalt to or greater than 25% degradation by Day 14 for the test substance to be considered as non-inhibitory. The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%. The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/L medium. The IC content of the test substance suspension in the mineral medium at the beginning of the test should be < 5% of the TC.

Reference substance:

benzoic acid, sodium salt

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 28.73 mg/L and therefore satisfied the validation criterion given in the OECD test guideline. The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD test guidelines. 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 OECD test guidelines.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

115

Sampling time:

28 d

Details on results:

Inorganic carbon values for the test substance, reference substance, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference substances and the toxicity control are given in Table. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on d 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5. Observations made on the contents of the test vessels are given in Table 6. Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test substance. The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of control replicate R1, reference substance replicate R1 and test substance 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. The test substance attained 115% degradation after 28 d and satisfied the 10-D window validation criterion, whereby 60% degradation must be attained within 10 d of the degradation exceeding 10%. The test substance can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. Degradation values in excess of 100% were considered to be due to an increase in the numbers of viable micro-organisms in the test substance vessels as a result of the readily biodegradable nature of the test substance. This effect occurs due to the micro-organisms utilizing the test substance as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100%.The toxicity control attained 53% degradation after 14 d thereby confirming that the test substance was not toxic to the sewage treatment micro-organisms used in the test. Variation in degradation rates on different sampling d was considered to be due to normal biological variation in CO2 evolution and/or sampling/analytical variation between the control and toxicity control vessels.

Results with reference substance:

Sodium benzoate attained 70% degradation after 14 d and 77% degradation after 28 d thereby confirming the suitability of the inoculum and test conditions. Variation in degradation rates on different sampling day was considered to be due to normal biological variation in CO2 evolution and/or sampling/analytical variation between the control and test substance vessels. Analysis of the test media taken from the reference substance culture vessels on day 0 and 28 for Dissolved Organic Carbon (DOC), (see Table 4), gave percentage degradation values of 100% 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.

Table 1: 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	0.70	0.70	1.05	0.70	0.70	0.82	1.05	0.70	0.70	0.70	0.70	0.70	0.70	0.35*
3	5.22	-	5.33	-	19.37	-	24.59	-	12.29	-	14.73	-	28.30	-
6	9.57	-	13.61	-	30.68	-	31.14	-	20.41	-	19.84	-	40.71**	-
8	12.61	-	16.40	-	35.55	-	44.38	-	27.86	-	29.35	-	49.54	-
10	11.97	-	16.07	-	33.29	-	40.47	-	30.21	-	30.78	-	49.02	-
14	18.59	-	18.93	-	35.70	-	43.86	-	40.91	-	33.55	-	51.34	-
21	21.07	-	21.41	-	39.55	-	44.84**	-	57.35	-	44.17**	-	-	-
28	23.63	-	23.41	-	43.45	-	45.25	-	62.27	-	52.42**	-	-	-
29	23.71	2.55	22.38	2.44	44.65	4.52	45.20	2.78	60.79	2.78	55.67	2.78	-	-

R₁– R₂= Replicates 1 and 2

Abs= CO₂absorber vessels

- = No value determined

*Sample analysed as no result was obtained from the original sample due to the instrument stopping mid sample

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

  

Table 2: Percentage biodegradation values

Day	% Degradation Sodium Benzoate	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
3	56	27	38
6	64	28	48
8	85	46	58
10	76	53	57
14	70	60	53
21	70	96	-
28	69	109	-
29*	77	115	-

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

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

Table 3: 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	9.53	-0.06	0
Sodium Benzoate 10 mg C/L R2	9.48	-0.23	0
Test Item 10.3 mg C/L R1	10.57**	0.26	2
Test Item 10.3 mg C/L R2	11.19**	-0.56	0
Test Item plus Sodium Benzoate Toxicity Control 20.3 mg C/L	20.29**	-0.10	0

  R₁– R₂= Replicates 1 and 2

*Corrected for control values. Negative values are due toasured 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 4: 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	9.60	96	<control	0	100
Sodium Benzoate 10 mg C/L R2	9.71	97	<control	0	100

 

R₁– R₂= Replicates 1 and 2

*Corrected for control values.

 

 

 

Table 5: pH values of the test preparations on Day 28

Test Vessel	pH
ControlR1	7.6
Control R2	7.6
Sodium Benzoate 10 mg C/L R1	7.5
Sodium Benzoate 10 mg C/L R2	7.5
Test Item 10.3 mg C/L R1	7.4
Test Item 10.3 mg C/L R2	7.5

 

R₁– R₂= Replicates 1 and 2 

Table 6: Observations on the test preparations throughout the test period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 10	Day 17	Day 24
Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Reference Item	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	R2	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
Toxicity Control		Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	-	-

R₁– R₂= Replicates 1 and 2

-= No observations made due to toxicity control being terminated after 14 d

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, the test substance was considered to be readily biodegradable (115% degradation within 28 d).

Executive summary:

A study was conducted to assess the ready biodegradability of the read across substance, Phosphoric acid, mono- and di-C6 -10 -alkyl ester, in an aerobic aqueous medium, according to OECD Guideline 301 B, "Ready Biodegradability; CO2 Evolution Test", EU Method C.4-C and OPPTS 835.3110 (Paragraph (M)) in compliance with GLP. The test substance, at a concentration of 10.3 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. The degradation of the test substance was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The test substance attained 115% degradation after 28 d. Degradation values in excess of 100% were considered to be due to an increase in the numbers of viable micro-organisms in the test substance vessels as a result of the readily biodegradable nature of the test substance. This effect occurs due to the micro-organisms utilizing the test substance as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100%. Under the test conditions, the test substance was considered to be readily biodegradable (115% degradation within 28 d) (Clarke, 2012).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

A study was conducted to assess the ready biodegradability of the read across substance, Phosphoric acid, mono- and di-C6 -10 -alkyl esters, in an aerobic aqueous medium, according to OECD Guideline 301 B, "Ready Biodegradability; CO2 Evolution Test", EU Method C.4-C and OPPTS 835.3110 (Paragraph (M)) in compliance with GLP. The test substance, at a concentration of 10.3 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. The degradation of the test substance was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The test substance attained 115% degradation after 28 d. Degradation values in excess of 100% were considered to be due to an increase in the numbers of viable micro-organisms in the test substance vessels as a result of the readily biodegradable nature of the test substance. This effect occurs due to the micro-organisms utilizing the test substance as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100%. Under the test conditions, the test substance was considered to be readily biodegradable (115% degradation within 28 d) (Clarke, 2012).