Phosphoric acid, 2-ethylhexyl ester

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:

key study

Study period:

The study was conducted between 24 May 2011 and 22 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method C.4-C of Commission Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):Not applicable.

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

Test SpeciesA mixed population of activated sewage sludge micro-organisms was obtained on 23 May 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 inoculumThe 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 3.2 g/l prior to use.* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an ovenEach 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.:

10 other: mg Carbon/l

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Pre-Study Solubility WorkData supplied by the Sponsor indicated that the test item was insoluble in water. Therefore pre-study solubility/dispersibility work was conducted to confirm this and to ascertain the method of preparation that would give the best testable dispersion (see details in any other information on materials and methods including tables section).Experimental PreparationFollowing results of the pre-study solubility work (see preliminary solubility work in any other information on materials and methods including tables section) the test item was dispersed into the test system with the aid of high shear mixing.An amount of test item (55.8 mg) was dispersed in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm) for approximately 15 minutes prior to dispersal in inoculated culture medium. The volume was adjusted to 3 litres to give a final concentration of 18.6 mg/l, equivalent to 10 mg carbon/l.A test concentration of 10 mg carbon/l was employed in the test following the recommendations of the Test Guideline.Reference ItemFor the purposes of the test, a reference item, sodium benzoate (C6H5COONa) (Sigma Aldrich Lot No MKBC1469), 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 ControlFor 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 (55.8 mg) was dispersed in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm) for approximately 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 18.6 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.Culture mediumThe culture medium used in this study (see culture medium details in any other information on materials and methods including tables section) was that recommended in the OECD Guidelines.Preparation of test systemThe 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 item, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.d) The test item plus the reference item 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 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 28.1 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) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 16, 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.The samples taken on Days 0, 2, 6, 8, 10, 14, 16, 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 (Na2CO3). Each analysis was carried out in triplicate.Dissolved organic carbon (DOC) analysis:Samples (30 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 0.45 µm Gelman AcroCap filters (approximately 5 ml discarded) prior to 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 (30 ml) were removed from the control and reference item 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-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 680DegC 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 measurementsThe 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.Evaluation of DataPlease see attached evaluation of data (calculation of carbon content, percentage degradation) in backgorund material section. Validation criteriaThe results of the degradation test are considered valid if in the same test the reference item yields equal to or greater than 60% degradation by Day 14.The test item may be considered to be readily biodegradable if eqaul to or greater than 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.The toxicity control (test item and sodium benzoate) should attain equalt to or greater than 25% degradation by Day 14 for the test item 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 item suspension in the mineral medium at the beginning of the test should be < 5% of the TC.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

None

Test performance:

The test satisfied the validation criteria.

Parameter:

% degradation (CO2 evolution)

Value:

98

Sampling time:

28 d

Details on results:

The test item attained 98% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B 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 must be attained within 10 days of the degradation exceeding 10%. However, the test item has exhibited the potential for rapid degradation.

Results with reference substance:

Sodium benzoate attained 111% degradation after 14 days and 112% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% and variation in the degradation values between different sampling days was considered to be due to sampling/analytical variation. 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 in any other information on materials and methods including tables section), gave percentage degradation values of 100% for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis.

Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1 (see attached background material). 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 perford on Days 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.

The total CO₂evolution in the control vessels on Day 28 was 18.63 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.

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.

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 CO₂detected 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 item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of test item replicate R₁. 

Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO₂into the second absorber vessels occurred.

The toxicity control attained 63% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. 

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% for Replicates R1 and R2. The degradation rates calculated from the

results of the DOC analyses were similar to those calculated from inorganic carbon analysis.

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	0.93	1.52	1.28	1.28	1.40	1.05	1.05	1.17	1.05	1.75	1.75	1.28	1.17	1.40
2	2.90	-	4.52	-	17.51	-	8.81	-	4.87	-	7.07	-	23.43	-
6	7.50	-	7.73	-	33.56	-	12.34	-	16.03	-	16.84	-	40.94	-
8	9.40	-	11.47	-	35.66	-	33.48	-	18.92	-	19.03	-	42.66	-
10	10.15	-	12.42	-	39.44	-	39.56	-	19.84	-	20.29	-	42.52	-
14	14.62	-	16.32	-	48.17	-	49.53	-	33.55	-	31.05	-	53.04	-
16	14.31	-	15.55	-	41.01	-	42.36	-	33.35	-	34.59	-	-	-
21	15.12	-	14.22	-	45.25	-	45.59	-	38.75	-	38.41	-	-	-
28	14.36	-	16.14	-	46.43*	-	46.76*	-	49.99	-	42.75*	-	-	-
29	18.15	2.78	17.60	3.37	51.24	2.55	51.57	3.02	47.25	2.32	47.03	2.32	-	-

R₁– R₂= Replicates 1 and 2

Abs= CO₂absorber vessels

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

- = No value determined

Table 2              Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
2	32	8	33
6	51	29	56
8	80	28	54
10	94	29	52
14	111	56	63
16	89	63	-
21	103	80	-
28	105	104	-
29*	112	98	-

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

*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	9.91	0.04	0
Sodium Benzoate 10 mg C/l R2	9.66	-0.17	0
Test Item 10 mg C/l R1	9.86**	-0.35	0
Test Item 10 mg C/l R2	9.89**	-0.16	0
Test Item plus Sodium Benzoate Toxicity Control 20 mg C/l	19.84**	-0.42	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 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	9.87	99	0.01	0	100
Sodium Benzoate 10 mg C/l R2	9.83	98	<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	7.5
Control R2	7.6
Sodium Benzoate 10 mg C/l R1	7.6
Sodium Benzoate 10 mg C/l R2	7.6
Test Item 10 mg C/l R1	7.4
Test Item 10 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 13	Day 20	Day 27
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 days

Validity criteria fulfilled:

yes

Remarks:

see any other information on results including tables section

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test item attained 98% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B 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 must be attained within 10 days of the degradation exceeding 10%. However, the test item has exhibited the potential for rapid degradation.

Executive summary:

Introduction. A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with 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.

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 98% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B 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 must be attained within 10 days of the degradation exceeding 10%. However, the test item has exhibited the potential for rapid degradation.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to GLP and according to a recognised testing guideline. However the experimental conduct was not entirely satisfactory given that there were an insufficient number of samples taken during the first few days of the test and hence the time at which 10% degredation had occured was missed. This resulted in the author extrapolating to the 10% trigger.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge was received from the municipal treatment plant of Breisgauer Bucht with a concentration corresponding to 30 mg dry soilids/L. The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in aerated tap water.

Duration of test (contact time):

28 d

Details on study design:

2 L gas wash bottles were used as reaction vessels. Seven of these were used for the test: two contained the test item; two contained innoculum only (blanks); two contained the reference compound; one contained the test item and the reference compound (toxicity control). Each of these was connected to two gas wash bottles in series, each filled with 200 mL 0.2 M NaOH. These were used to collect the CO2 produced in the reactor vessels.8.7 mL of activated sludge were added to the test vessels and 1491.3 mL mineral medium were added to give a final volume of 1500 mL. The system was sealed and aerated with C02-free air at a rate of 50-100 mL/min overnight (2.7 - 5.5 bubbles / second). The CO2 absorber wash bottles were filled with 0.2 M NaOH. 55.2 and 55.4 mg of test item (corresponding to concentrations of 20.1 and 20.2 mg?L organic carbon). 5.15 mL of a stock solution of 10 g/L sodium benzoate (corresponding to 20 mg/L or organic carbon) were added to the reference vessels. The test was thus started. The test system was incubated in diffuse light at 21 to 22.5°C. At the beginning of the study the inorganic carbon (IC) concentration of the 0.2 M NaOH used for the C02- absorbtion flasks was determined as 0.2 ppm. On the 4th, 7th, 11th, 14th, 21st and 28th day, 4 mL NaOH from the first of two C02-absorber flasks connected in line and the IC's were determined. On the 28th day 1 mL concentrated hydrogen chloride acid (HCI) was added into each reactor to release the C02 dissolved in water and the IC was determined in both C02- absorber flasks in line.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

70.8

Sampling time:

28 d

Results with reference substance:

The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days.

Table 1. Ultimate biodegradation after x days (% of theoretical CO2)

Reactor	Day	0	4	7	11	14	21	28	28 (after acidification)
5	Test flask	0	19.5	27.2	43.2	57.8	64.2	71.7	66.0
6	Test flask	0	18.6	25.8	59.0	72.3	77.7	78.4	75.5
11	Reference flask	0	81.0	93.6	93.5	96.5	96.2	101.4	100.4
12	Reference flask	0	81.5	90.7	91.6	94.4	89.9	89.2	89.2
8	Inhibitioon control Test itemReference item	0	49.8	60.1	64.4	83.5	85.3	86.0	86.2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Servoxyl VPTZ 100 was determined to be readily biodegradable (but failing 10-day window).

Executive summary:

A 28 day biodegradability test was conducted according to OECD 301 B (CO2 evolution test) with Servoxyl VPTZ 100. The mean degradation extent of the test item was 70.8% within 28 days after acidification. The substance is considered to be readily biodegradable (but failing 10 -day window).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.07.2004 - 13.08.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with an internationally accepted guideline, and in accordance with the principles of GLP.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

150 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

aniline

Parameter:

other: ThOD

Value:

77

Sampling time:

28 d

Results with reference substance:

After 14 days the reference cpompund was sufficiently degraded to 91 % of the ThOD, thus confirming the suitability of the used activated sludge inoculum and fulfilling the validity criteria mentionend in the relevant test Guideline. It was degraded to 110 % of the ThOD after 28 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test substance can considered to be readily biodegradable, but failling the 10-day window.

Executive summary:

The test item was tested in a manometric respirometry test for 28 days to determine whether the test substance is readily biodegradable.

 

The concentration tested was 70 mg per liter in duplicate, corresponding to a Theoretical Oxygen demand (ThOD) of 150 mg/L.

 

As reference compound (procedure control) Aniline in a concentration of 60 mg/L, corresponding to 144 mg/L ThOD was used.

 

After 14 days the reference compound was sufficiently degraded to 91 % of the ThOD thus confirming the suitability of the used activated sludge inoculum and fulfilling the validity criteria mentioned in the relevant test guideline. It was degraded to 110 % of the ThOD after 28 days.

 

At the end of the exposure period degradation rates of 77 and 75 % of the theoretical Oxygen Demand (ThOD) of the test substance were measured. During a 10 days window a degradation rate of at least 60 % which is required by the OECD Guideline to qualify for readily biodegradation was not reached.

 

However, in less than 28 days a degradation rate of more than 60 % was determined. The test substance can be considered as biodegradable under test conditions.

 

The biodegradation in the toxicity control containing both the test substance and the reference compound in the same concentrations as listed above shows a similar course of degradation compared to the procedure control. After 14 days a degradation rate of 51 % was recorded, rising to 71 % after 28 days (i.e. approx. 105 % and 148 % corresponding to the reference compound), thus demonstrating that the test substance does not inhibit the microorganisms.

 

For determination of possible abiotic degradation of the test substance an “abiotic control” was tested, containing the test substance and 3,5-Dichlorophenole in a sufficient concentration to inhibit microorganisms and thus prevent biological degradation. After 28 days of exposure virtually no degradation was recorded.

Description of key information

Readily biodegradable, but failing 10-day window; Clarke (2010); OECD 301B (CO2 Evolution Test)
Readily biodegradable, but failing 10-day window; Aventis (2004); OECD 301F (Manometric Respirometry Test)
Readily biodegradable; Brunswiik-Titze (2005); OECD 301B (CO2 Evolution Test)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Three key studies were identified, where the test item was assessed for it's ready biodegradability in accordance with internationally recognised OECD methods (OECD 301B and OECD 301F).

Although considered a valid study, Brunswiik-Titze (2005) extrapolated the beginning of the 10 -day window based on day 0 (0 % degradation) and day 4 (19 % degradation) to conclude that the 10 -day window had been reached. This assumption is subject to considerable error, especially considering the day 14 biodegradation was only 65 %, and as such, this study was not used in the application of the biodegradability criteria.

The studies by Clarke (2010) and Aventis (2004), assessed the ready biodegradability of the test item using OECD methods 301B and 301F respectively. These studies were comparable, in that they both led to the conclusion that the test item was readily biodegradable, but failing the 10 -day window.

The studies by Clarke (2010) and Aventis (2004) were used as the conclusive evidence for the biodegradation in water: screening tests endpoint.

Ready Biodegradability of Multi-Constituent Substances

For the purpose of assessing the ready biodegradability of multi-constituent substances using standard screening tests, the 10 -day window should not be considered, as it can only be interpreted correctly if a normal S-shaped degradation pattern is observed (UN/SCEGHS/15/INF.35, 2008). For multi-constituent substances, sequential biodegradation of the individual structures can take place, and in such cases, the 10 -day window is a poor indicator of the biodegradation kinetics and should not be applied to interpret the results of the test. The studies by Clarke (2010) and Aventis (2004) clearly indicate two phases of degradation.

The following criterion is therefore used to assess the biodegradability of phosphoric acid, 2 -ethylhexyl ester (

UN/SCEGHS/15/INF.35, 2008):

"Multi-constituent substances do not need to fulfil the specified levels of biodegradation within a 10 days of the start of biodegradation. Such samples surpassing the 60 % limit value within the standard test duration of 28 days are considered as readily biodegradable substances".

Phosphoric acid, 2 -ethylhexyl ester is therefore considered a readily biodegradable multi-constituent substance.