1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-nortall-oil 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:

The study was conducted between 10 November 2011 and 10 January 2012.

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 with the exception below. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The test duration was extended up to 60 days . The test volume employed was increased from 3 litres to 4 litres.The concentration of inoculum used was increased from 30 mg suspended solids (ss)/l to 50 mg ss/l.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The test duration was extended up to 60 days . The test volume employed was increased from 3 litres to 4 litres.The concentration of inoculum used was increased from 30 mg suspended solids (ss)/l to 50 mg ss/l.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

yes

Remarks:

The test duration was extended up to 60 days . The test volume employed was increased from 3 litres to 4 litres.The concentration of inoculum used was increased from 30 mg suspended solids (ss)/l to 50 mg ss/l.

Principles of method if other than guideline:

Following the recommendations of the ECHA REACH Guidance Document “Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance” the following modifications to a standard OECD 301B test were made:
Test duration: The test duration may be extended up to 60 days dependent on the level of biodegradation observed.
Testing in larger vessels: The test volume employed was increased from 3 litres to 4 litres.
Increasing the biomass: The concentration of inoculum used was increased from 30 mg suspended solids (ss)/l to 50 mg ss/l.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test Species:
A mixed population of activated sewage sludge micro-organisms was obtained on 9 November 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 medium:
The culture medium used in this study was that recommended in the OECD Guidelines.

Culture Medium
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l

pH = 7.4

Solution b CaCl2 27.50 g/l
Solution c MgSO4.7H2O 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

Duration of test (contact time):

60 d

Initial conc.:

7.4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Pre-Study Solubility Work:
Information supplied by the Sponsor indicated that the test item was partially soluble in water. Therefore pre-study solubility/dispersibility work was performed in order to determine the most suitable method of preparation (see Appendix 2 in any other information on results incl. tables section).

Experimental Preparation::
For the purpose of the test, the test item was dispersed directly in culture medium with the aid of high shear mixing.
An amount of test item (29.6 mg) was dispersed in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm for 15 minutes) prior to dispersal in inoculated culture medium. The volume was adjusted to 4 litres to give a final concentration of 7.4 mg/l, equivalent to 5 mg carbon/l.

Results obtained from the Assessment of the Inhibitory Effect on the Respiration of Activated Sewage Sludge (Harlan project number: 41101677) where the EC50 value for the test item was 97 mg/l (95% confidence limits 79 – 120 mg/l) suggested that the test item may exhibit some inhibitory effect on the inoculum at the test concentration of 10 mg carbon/l, so therefore the test concentration employed in the study was reduced to 5 mg C/l (equivalent to 7.4 mg test item/l).

Reference Item:
For 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 15 minutes. An aliquot (68.4 ml) of this stock solution was added to the test vessel containing inoculated culture medium and the volume adjusted to 4 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 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 (29.6 mg) was dispersed in approximately 400 ml of culture medium with the aid of high shear mixing (7500 rpm for 15 minutes) prior to dispersal in inoculated culture medium. An aliquot (68.4 ml) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 4 litres to give a final concentration of 7.4 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 15 mg carbon/l.

Preparation of test system:
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 4 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 5 mg carbon/l.
d) The test item plus the reference item in inoculated culture medium to give a final concentration of 15 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 50 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21DegC, 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 58.8 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 4 litres by the addition of culture medium.

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/minute where necessary.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
T
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 and analysis:

CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessels on Days 0, 7, 14, 21, 28, 35, 42, 49, 56, 60 and 61. The second absorber vessel was sampled on Days 0 and 61.
The samples taken on Days 0, 7, 14, 21, 28, 35, 42, 49, 56, 60 and 61 were analysed for CO2 immediately.
On Day 60, 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 61.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser. Samples (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 60 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-LCSH TOC analyser and a Shimadzu TOC-VCPH TOC analyser. Samples (50 or 800 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 68degC 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 60, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Information supplied by the Sponsor indicated that the test item was partially soluble in water. Therefore pre-study solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test performance:

The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3 in any other on results including tables section) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guideline.
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 Guideline.

Parameter:

% degradation (CO2 evolution)

Value:

31

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

77

Sampling time:

60 d

Details on results:

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 (attached in background materila section). 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 Days 0 and 60 are given in Table 4. The pH values of the test preparations on Day 60 are given in Table 5. Observations made on the test vessels throughout the study period are given in Table 6.
All tables are in any other information on results including tables section.

Acidification of the test vessels on Day 60 followed by the final analyses on Day 61 was conducted according to the methods specified in the Test Guideline. 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 61 samples originated from dissolved CO2 that was present in the test vessels on Day 60 and hence the biodegradation value calculated from the Day 61 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 61 showed an increase in all replicate vessels with exception of control replicate R1 and the toxicity control. Inorganic carbon analysis of the samples from the second absorber vessels on Day 61 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
T
he test item attained 31% degradation after 28 days and 77% degradation after 60 days.

The toxicity control attained 69% degradation after 14 days, 88% degradation after 28 days and 81% degradation after 60 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. This slight decrease in degradation between Days 28 and 60 was considered to be due to sampling/analytical variation.

Results with reference substance:

Sodium benzoate attained 103% degradation after 14 days, 87% degradation after 28 days and 101% degradation after 60 days thereby confirming the suitability of the inoculum and test conditions. This slight decrease in degradation between Days 14, 28 and 60 and degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Analysis of the test media taken from the reference item culture vessels on Days 0 and 60 for Dissolved Organic Carbon (DOC), (see Table 4 in any other information on results including tables section), gave percentage degradation values of 98% and 100% respectively 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.

Appendix 2      Pre-Study Solubility Work

Data supplied by the Sponsor indicated that the test item was partially soluble in water, therefore the following pre-study solubility/dispersibility work was conducted. 

(i)           Ultrasonication: An amount of test item (100 mg) was dispersed in 1 litre of deionised reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute prior to ultrasonication for 30 minutes. This formed a cloudy dispersion.   

This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test. 

(ii)         Ultrasonication: An amount of test item (50 mg) was dispersed in approximately 450 ml of culture media with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 3 litres. This formed a very slightly cloudy dispersion with a small amount of foam on surface and no undissolved test item visible. After magnetic stirring for 24 hours an oily slick of test item was visible on the surface of a slightly cloudy dispersion.

(iii)        High shear mixing: An amount of test item (50 mg) was dispersed in approximately 450 ml of culture media with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The volume was then adjusted to a final volume of 3 litres. This formed a clear colourless media column with foam on surface and no undissolved test item visible. After magnetic stirring for 68 hours the appearance remained unchanged.

(iv)        High shear mixing using an inert carrier, silica gel (an approved inert carrier, ISO 1995 and in the published literature (Handleyet al, 2002)). An amount of test item (50 mg) was adsorbed onto silica gel (100 mg) and dispersed inapproximately 450 ml of culture media with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The volume was then adjusted to a final volume of 3 litres. This formed a clear colourless media column with foam

 on surface and adhered to the sides of the vessel. After 48 hours magnetic stirring white particles of test item were visible on the surface in a clear colourless media column.

(v)           Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fibre filter paper was attempted.

An amount of test item (1000 mg) was dissolved, with the aid of shaking by hand for approximately 30 seconds, in acetone (5 ml) and formed an orange/brown solution. An aliquot (225 µl) of this solvent stock solution was dispensed to GF/A filter paper. The solvent was allowed to evaporate to dryness for approximately 30 minutes prior to addition of the test item coated filter paper to approximately 450 ml culture medium and being subjected to high shear mixing (approximately 7500 rpm, 5 minutes). The volume was then adjusted to 3 litres culture medium and formed a clear colourless media column with broken up filter paper dispersed throughout and foam on surface. After 24 hours magnetic stirring an oily layer of test item was visible on the surface of a cloudy dispersion containing broken up pieces of filter paper.

(vi)         Addition onto solid supports: The use of a glass microscope slide and glass fibre filter papers was attempted.

When using a glass microscope slide, an amount of test item (50 mg) was weighed onto a glass slide and added to 3 litres of culture medium. This formed a clear colourless media column with the glass slide visible on the bottom of the vessel with the test item adhered to it and floating on the surafce. 

When using GF/A filter paper, an amount of test item (50 mg) was weighed onto a GF/A filter paper and added to 3 litres of culture media. This formed a clear colourless water column with the test item visible adhered to the filter paper floating on the surface. After 24 hours magnetic stirring test item was visible on the surface of a cloudy dispersion containing broken up pieces of filter paper.

(vii)           Preliminary Solution in a Non-Volatile, Non-Degradable Solvent:An amount of test item (100 mg) was dispersed in 5 ml of silicone oil with the aid of shaking by hand for approximately 30 seconds followed by ultrasonication for 15 minutes. This formed a pale yellow dispersion with a orange globules of test item visible throughout.

From the pre-study solubility work it was concluded that the best method of preparation was by preparing by dispersing the test item into the test system with the aid of high shear mixing and then addition to inoculated culture medium as this method appeared to increase the dispersibility of the test item over the other methods.

 

Table1              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	2.10	1.40	1.17		1.28	1.40	1.17	1.28	1.17	1.17	1.05	1.05	1.17	1.05	1.28
7	12.99	-	11.60		-	46.17	-	48.14	-	10.33	-	9.98	-	48.49	-
14	17.19	-	17.30		-	57.44	-	59.51	-	16.72	-	16.38	-	58.59	-
21	20.30	-	19.38		-	61.69	-	65.70	-	23.97	-	23.51	-	66.51	-
28	21.32	-	22.80		-	55.52	-	58.48	-	28.73	-	27.93	-	74.79	-
35	23.46	-	23.69		-	54.97	-	51.34	-	31.17	-	32.07	-	75.59	-
42	24.67	-	24.00		-	59.04	-	60.16	-	34.03	-	34.14	-	73.12	-
49	27.22	-	26.65		-	59.25	-	60.82	-	38.87	-	35.05	-	72.80	-
56	27.72	-	28.72		-	69.70	-	57.78	-	37.41	-	40.97	-	79.60	-
60	33.86	-	32.65		-	69.28	-	74.92	-	46.37	-	46.92	-	86.65	-
61	33.66	2.90	33.11		2.78	71.28	4.52	75.02	2.55	48.18	4.52	47.41	3.02	81.73	2.55

R₁– R₂= Replicates 1 and 2

Abs= CO₂absorber vessels

- = No value determined

Table2              Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
7	87	0	60
14	103	0	69
21	110	20	78
28	87	31	88
35	74	40	87
42	88	49	81
49	83	50	76
56	89	55	86
60	97	67	89
61*	101	77	81

*Day 61 values corrected to include any carry-over of CO₂detected in Absorber 2

Table3              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	8.44	0.00	0
Sodium Benzoate 10 mg C/l R2	9.60	0.01	0
Test Item 5 mg C/l R1	5.75**	0.01	0
Test Item 5 mg C/l R2	5.31**	0.00	0
Test Item plus Sodium Benzoate Toxicity Control 15 mg C/l	16.16**	0.01	0

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

Table4              Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 60

Test Vessel	DOC*Concentration
	Day 0		Day 60
	mg C/l	% of Nominal Carbon Content	mg C/l	% of Initial Carbon Concentration	% Degradation
Sodium Benzoate 10 mg C/l R1	8.45	85	0.21	2	98
Sodium Benzoate 10 mg C/l R2	9.60	96	0.01	0	100

R₁– R₂= Replicates 1 and 2

*Corrected for control values.

Table5              pH Values of the Test Preparations on Day 60

Test Vessel	pH
ControlR1	7.6
Control R2	7.6
Sodium Benzoate 10 mg C/l R1	7.7
Sodium Benzoate 10 mg C/l R2	7.8
Test Item 5 mg C/l R1	7.8
Test Item 5 mg C/l R2	7.7
Toxicity Control	7.7

R₁– R₂= Replicates 1 and 2

Table6              Observations on the Test Preparations Throughout the Test Period

 

Test Vessel		Observations on Test Preparations
		Day 0	Day 7	Day 14	Day 21	Day 28
Control	R1	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion
	R2	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion
Reference Item	R1	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible
	R2	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible
Test Item	R1	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible
	R2	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible
Toxicity Control		Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible

R₁– R₂= Replicates 1 and 2

Table 6 (continued)          Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 35	Day 42	Day 49	Day 56
Control	R1	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion
	R2	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion	Light brown cloudy dispersion
Reference Item	R1	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible
	R2	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible	Light brown cloudy dispersion, no undissolved reference item visible
Test Item	R1	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible
	R2	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible	Light brown cloudy dispersion, no undissolved test item visible
Toxicity Control		Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible	Light brown cloudy dispersion, no undissolved test or reference item visible

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

other: The test item attained 31% degradation after 28 days and 77% degradation after 60 days.

Conclusions:

The test item attained 31% degradation after 28 days and 77% degradation after 60 days.

Executive summary:

Introduction.

A study was performed to assess the biodegradability of the test item in an aerobic aqueous medium. The test method was designed to follow 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)). However following the recommendations of the ECHA REACH Guidance Document “Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance” the following modifications to a standard OECD 301B test were made:

Test duration:

The test duration may be extended up to 60 days dependent on the level of biodegradation observed.

Testing in larger vessels: The test volume employed was increased from 3 litres to 4 litres.

Increasing the biomass: The concentration of inoculum used was increased from 30 mg suspended solids (ss)/l to 50 mg ss/l.

Methods.

The test item, at a concentration of 5 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 60 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 and Conclusion.

The test item attained 31% degradation after 28 days and 77% degradation after 60 days.

Description of key information

In a test according to OECD 301B, the substance attained 31% degradation after 28 days and 77% degradation after 60 days.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater