Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 2 March 2010 and 01 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

SULFOCLOR LF 1013

IUPAC Name:

SULFOCLOR LF 1013

Details on test material:

Sponsor's identification :SULFOCLOR LF 1013
Description :pale yellow slightly viscous liquid
Batch number :not supplied
Date received :5 February 2010
Expiry date :not supplied
Storage conditions :room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the SULFOCLOR LF 1013 is the responsibility of the Sponsor. The chemical specification of the test item provided by the Sponsor is presented in Appendix 1 (see in attached section).
Chemical Composition
Further to the chemical specification presented in Appendix 1 (see in attached section), the Sponsor provided the following chemical composition for the test item:
Sulphochlorinated C14 to C17 linear paraffin
CnHxCly(SO2Cl)z n = 14 to 17, x = 2n + 2 – y – z

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn trent Water Plc
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.1 g/l prior to use.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel.

The culture medium used in this study (see Appendix 2 See in other information on materials and methods section) was that recommended in the OECD Guidelines.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

18.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 1 March 2010 from the aeration stage of the Severn Trent Water Plcsewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Pre-Study Solubility Work
The Sponsor did not provide any water solubility data for the test item. Therefore the following pre-study solubility/dispersibility work was conducted:
(i) Ultrasonication: An amount of test item (50 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 approximately 30 minutes. This formed a cloudy dispersion with an oily slick of test item visible on the surface.
(ii) High shear mixing: An amount of test item (50 mg) was dispersed in 1 litre of deionised reverse osmosis purified water and subjected to high shear mixing at approximately 7500 rpm for approximately 30 minutes. This formed a cloudy dispersion with a few small globules of test item visible on the surface.
This work confirmed that the test item was poorly soluble in water. Therefore the following additional solubility work was conducted to ascertain the method that would give the best testable dispersion:
(iii) Ultrasonication: An amount of test item (50 mg) was dispersed in 500 ml of deionised reverse osmosis purified water with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 3 litres. This formed a cloudy dispersion with an oily slick of test item on the surface.
(iv) High shear mixing: An amount of test item (50 mg) was dispersed in 500 ml of deionised reverse osmosis purified water with the aid of high shear mixing at approximately 7500 rpm for 10 minutes. The volume was then adjusted to a final volume of 3 litres. This formed a cloudy dispersion with a few small globules of test item visible on the surface.
(v) High shear mixing using an inert carrier, silica gel (an approved inert carrier, ISO 1995 and in the published literature (Handley et al, 2002)). An amount of test item (50 mg) was adsorbed onto silica gel (100 mg) and dispersed in approximately 500 ml of deionised reverse osmosis purified water with the aid of high shear mixing at approximately 7500 rpm for 10 minutes. The volume was then adjusted to a final volume of 3 litres. This formed a cloudy dispersion with no undissolved test item visible.

A volatility check was performed on the test item by passing a steady stream of compressed air over a known weight of test item for 3 hours as high shear mixing cannot be performed on volatile test materials. This resulted in 2% loss of test item over this time so the test item was considered not to be volatile. As such it was concluded appropriate to disperse the test item with the aid of high shear mixing and silica gel as this method appeared to be the most effective in increasing its the dispersibility.

Experimental Preparation
Following the recommendations of the International Standards Organisation (ISO 1995) and in the published literature (Handley et al, 2002) the test item was adsorbed onto silica gel prior to 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 (56.7 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No 101K3700) prior to dispersal in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm, 10 minutes). The test item/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 18.9 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.
Control vessels were prepared containing 100 mg silica gel per 3 litres of inoculated culture medium in order to maintain consistency between the control and test item vessels.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the concentration, homogeneity or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

GLP COMPLIANCE STATEMENT
With the exception noted below the work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.
No analysis was carried out 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.
This report fully and accurately reflects the procedures used and data generated.

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 (56.7 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No.101K3700) prior to dispersal in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm, 10 minutes). The test item/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 3 litres to give a final concentration of 18.9 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 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 plus 100 mg silica gel.
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
c) The test item, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
d) The test item plus the reference item in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Silica gel was added to the control and reference item vessels in order to maintain consistency between these vessels and the test item vessels.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21°C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 ml of culture medium and 29.0 ml of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.
Sampling and analysis

CO2 analysis
Samples (2 ml) were taken from the control, standard and test item first CO2 absorber vessels on Days 0, 2, 7, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 7, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, standard and test item and on Day 0 for the toxicity control.
The samples taken on Days 0, 2, 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 standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
Dissolved organic carbon (DOC) analysis
Samples (20 ml) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through Gelman 0.45 µm AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.

DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
On Days 0 and 28 samples (20 ml) were removed from the control and reference item vessels and filtered through Gelman 0.45 µm AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-Vsch TOC analyser. Samples (27, 13 µl or 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 standard 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.

Evaluation of Data
Calculation of carbon content and Percentage degradation (see in attached section).

Validation criteria
The results of the degradation test are considered valid if in the same test the reference item yields > 60% degradation by Day 14.
The test item may be considered to be readily biodegradable if > 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 > 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

Reference substance:

other: sodium benzoate,

Results and discussion

Test performance:

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.
Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1 (see in other information on results section). Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 (see in other information on results section) and the biodegradation curves are presented in Figure 1 (see in attached section). Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3 (see in other information on results section), and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4 (see in other information on results section). The pH values of the test preparations on Day 28 are given in Table 5 (see in other information on results section).
The total CO2 evolution in the control vessels on Day 28 was 30.04 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.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

89

Sampling time:

28 d

Remarks on result:

other: St dev not stated

Details on results:

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

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. 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 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.
The total CO2 evolution in the control vessels on Day 28 was 30.04 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 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 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 material Replicate R1. 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.

SULFLOCLOR LF 1013 attained 89% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. SULFLOCLOR LF 1013 can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 71% degradation after 14 days and 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 97% 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.
Observations made on Day 0 of the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the reference item vessels were light brown dispersions with no undissolved reference item visible. The test item vessels were observed to be cloudy light brown dispersions with no undissolved test item visible. The contents of the toxicity control vessel was observed to be a cloudy light brown dispersion with no undissolved test or reference item visible.

BOD5 / COD results

Results with reference substance:

The reference item (Sodium benzoate) attained 99% degradation after 14 days and 105% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. 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 28 for Dissolved Organic Carbon (DOC), see Table 4 (see in any other information on results section), gave percentage degradation values of 97% 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.
Observations made on Day 0 of the test period (see Table 6 (see in any other information on results section) showed the contents of the control vessels to be light brown dispersions and the contents of the reference item vessels were light brown dispersions with no undissolved reference item visible. The test item vessels were observed to be cloudy light brown dispersions with no undissolved test item visible. The contents of the toxicity control vessel was observed to be a cloudy light brown dispersion with no undissolved test or reference item visible.

Any other information on results incl. tables

Table1              Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg IC)				Sodium Benzoate (mg IC)				Sulfoclor LF 1013 (mg IC)				Sulfoclor LF 1013 plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.40	1.40	1.63	1.40	1.05	1.05	1.40	0.93	1.17	0.58*	1.40	1.05	1.05	1.40
2	11.72	-	6.61	-	11.83	-	20.18	-	7.89	-	11.60	-	26.21	-
7	13.49*	-	16.49	-	28.26*	-	28.26*	-	26.64	-	26.76	-	34.71*	-
8	15.59	-	16.05	-	38.07	-	37.84	-	26.14	-	26.14	-	38.07	-
10	18.47	-	16.65	-	42.98	-	39.78	-	29.41	-	25.42	-	46.74	-
14	20.74	-	20.51	-	52.25	-	48.17	-	45.22	-	41.14	-	63.35	-
21	21.18	-	21.52	-	54.76*	-	49.46	-	49.46	-	45.07	-	-	-
28	24.53	-	24.64	-	56.67*	-	55.55	-	54.66*	-	47.49*	-	-	-
29	26.16	2.32	26.61*	2.44	59.45	2.44	56.56	2.09	54.44	2.32	51.66	2.32	-	-

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

R₁– R₂= Replicates 1 and 2

Table2              Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Sulfoclor LF 1013	% Degradation Sulfoclor LF 1013 plus Sodium Benzoate Toxicity Control
0	0	0	0
2	23	2	28
7	44	39	33
8	74	34	37
10	79	33	49
14	99	75	71
21	103	86	-
28	105	88	-
29*	105	89	-

-= 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

 

Abs= CO₂absorber vessels

- = No value determined

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	10.44	0.43	4
Sodium Benzoate 10 mg C/l R2	10.76 ^†	-0.25	0
Sulfoclor LF 1013 10 mg C/l R1	11.36**^†	-0.36	0
Sulfoclor LF 1013 10 mg C/l R2	9.46**	-1.22	0
Sulfoclor LF 1013 plus Sodium Benzoate Toxicity Control 20 mg C/l	19.84**	0.31	2

R₁– R₂= Replicates 1 and 2

*Corrected for control values. Negative values are due to asured 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

^† Duplicate sample analysed as the original sample result was deemed anomalous

Table4              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	10.00	100	0.35	4	97
Sodium Benzoate 10 mg C/l R2	11.01	110	0.32	3	97

R₁– R₂= Replicates 1 and 2

*Corrected for control values.

Table5              pH Values of the Test Preparations on Day 28

Test Vessel	pH
ControlR1	7.5
Control R2	7.5
Sodium Benzoate 10 mg C/l R1	7.5
Sodium Benzoate 10 mg C/l R2	7.5
Sulfoclor LF 1013 10 mg C/l R1	7.5
Sulfoclor LF 1013 10 mg C/l R2	7.5

 

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 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
Sulfoclor LF 1013	R1	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible
	R2	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible	Cloudy light brown dispersion. No undissolved test item visible
Toxicity Control		Cloudy light brown dispersion. No undissolved test or reference item visible	Cloudy light brown dispersion. No undissolved test or reference item visible	Cloudy light brown dispersion. No undissolved test or reference item visible	Cloudy light brown dispersion. No undissolved test or reference item visible	Cloudy light brown dispersion. No undissolved test or reference item visible

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

SULFLOCLOR LF 1013 attained 89% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. SULFOCLOR LF 1013 can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the SULFOCLOR LF 1013 in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO₂Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Methods.

SULFOCLOR LF 1013, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handleyet al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid its dispersion in the test medium and to increase the surface area of the test item exposed to the test organisms.

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

Results.

SULFOCLOR LF 1013 attained 89% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. SULFOCLOR LF 1013 can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Conclusion

SULFOCLOR LF 1013 attained 89% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. SULFOCLOR LF 1013 can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.