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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:

Study initiation: March 24, 2009; Study completion: August 7, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Chemicals Evaluation and Research Institute, Japan

- Concentration of sludge: Mixed liquor suspended solid (MLSS): 3300 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: The biological oxygen demand (BOD) was measured over a 28-day period. After this period, amounts of the dissolved organic carbon (DOC) and residual test susbtance were measured. The biodegradability of the test substance was evaluated from these results.

Details on study design:

Study conditions:
1) Temperature: 25 ± 1ºC
2) Exposure period: 28 days
3) Stirring: Continuous stirring with magnetic stirrer
4) Test volume: 300 mL
5) Concentration: test substance (bottles 3-6): 100 mg/l
Aniline (bottle 1) : 100 mg/l
Activated sludge (bottles 1-5): 30 mg/l

Test bottle contents:
Bottle 1: Activity control (aniline + activated sludge + basal medium)
29.5 μL (30.0 mg) of aniline was added to the basal medium, and then 2.7 ml of activated sludge was added.

Bottle 2: Inoculum blank (activated sludge + basal medium)
2.7 ml of activated sludge was added to the basal medium.

Bottles 3, 4, 5: Test suspensions (test substance + activated sludge + basal medium)
30.0 mg of the test substance was added to the basal medium then 2.7 ml activated sludge was added.

Bottle 6: Abiotic control (test substance + purified water)
30.0 mg of the test substance was added to 300 ml of purified water.

Reference substance:

aniline

Test performance:

There was no specific factor which might have affected the reliability of the test results.
The test was judged valid since the measurement results fulfilled all of the validity criteria of the guideline.

Parameter:

other: BOD

Value:

52

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

47

Sampling time:

28 d

Details on results:

Observation of the test solutions after exposure period:
The color of the test solutions and growth of the sludge were observed in contrast with the control (bottle 2).
The test solutions in bottle 1 and 5 were white, solutions in bottles 3, 4 and 6 were slightly white.
Growth of the sludge was observed in bottle 1, 3, 4 and 5.

pH measurement:
After 28 days of exposure, pH values were 6.9, 7.0, 7.0, 8.2 for bottles 3, 4, 5 and 6, respectively.

Degradability based on the BOD:
The BOD (maximum theoretical value = 103.7 mg) in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 49.2, 57.0 and 56.3 mg respectively, and the BOD in bottle 6 was 0.7 mg.
The degrability based on the BOD measurement was calculated to be 47, 55 and 54% for bottles 3, 4 and 5 respectively.

Degradability based on the DOC:
The DOC (initial amount 25.8 mg) in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 0.1, 0.0 and -0.2 mg respectively, and the DOC in bottle 6 was 1.7 mg.
The degradability based on the DOC was not calculated since the test substance was insoluble in water and the DOC in bottle 6 is less than 90% of initial amount.

Degradability based on the residual test substance amount:
The amounts of residual test substance (Initial amount = 30.0 mg) were 16.6, 14.9 and 15.3 mg in bottles 3, 4 and 5 respectively and 29.3 mg in bottle 6.
The degradabilities based on the residual test substance amount were calculated to be 43, 49 and 48% for bottles 3, 4, and 5, respectively.

From the degradability results based on the BOD (av. 52%) and the residual test substance amount (av. 47%), it is concluded that the test substance is not readily biodegradable under the conditions of this test.

Result tables attached as attached background material.

Results with reference substance:

The aniline control (Bottle 1) attained 73.0% degradability after 28 days.

From the degradability results based on the BOD (av. 52%) and the residual test substance amount (av. 47%), it is concluded that the test substance is not readily biodegradable under the conditions of this test.

To confirm existence of transformed product, HPLC with reverse-phase column analysis and LCIMS analysis were performed. Neither new peak on chromatogram nor molecular-related ion on Mass spectrum were detected. It is concluded that the test substance was not transformed structurally under the conditions of this test.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Not readily biodegradable.

Conclusions:

The test substance is not readily degradable. No transformed product was formed.

Executive summary:

A ready biodegradation test of 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' has been conducted, using the Japanese methodology for new substance notification, i.e. OECD 301C MITI (I) methodology. The method for introduction of the test substance into the test system was not described. The test substance concentration was 100 mg/l. There was no indication of steps being taken to minimise or prevent evaporation during test substance loading or during the test. The inoculum was a mixed liquor obtained from CERI, Japan; in accordance with the guideline the sludge should be non-adapted and whilst this is not explicitly stated, it is reasonable to assume that such an important criterion would have been met. The concentration of inoculum in the test system was 30 mg suspended solids/l. Assessment of biodegradation in both tests was made by BOD, analysed by closed system oxygen consumption analysis, supported by residual test substance analysis by gel permeation chromatography. DOC measurements were made but degradation was not assessed on this basis due to poor recoveries in the abiotic control, possibly due to the very low solubility of the test substance. At the end of the 28 d test period, the following results are reported: 47% - 55% degradation (based on BOD); 43% - 49% degradation (based on residual test substance). The kinetics appear to show that the degradation proceeded smoothly without significant plateaux in the curve. The study was conducted in accordance with GLP and is considered to be reliable, though the results are not necessarily meaningful for exposure assessment purposes. Distillates (Fischer-Tropsch), C18-50-branched, cyclic and linear is not readily biodegradable in this study.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-14 to 2013-02-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C30; additional chemical analysis performed during the study indicated that test material losses occurred due to a number of mechanisms, including abiotic losses e.g. from volatisilation or formation of non-extractable residues.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A mixed population of activated sludge micro-organisms was obtained on 14 January 2013 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 mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was them maintained on continuous aeration in the laboratory at a temperature of approximately 21°C 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-weighted GF/A filter paper using a Buchner funnel. Filtration was continued for a further three minutes after rinsing the filter three successive timeswith 10 mL of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C 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 3.0 g/L prior to use.

Abiotic samples were prepared by the autoclaving an aliquot of inoculum prior to addition to the test vessels. Additionally an aliquot (20 mL) of a 10 g/L aqueous solution of sodium azide was added to the test vessels to maintain sterility through the duration of the incubation period.

Duration of test (contact time):

29 d

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of test system
The following test preparations were prepared and inoculated in a 5 litre test culture each containing 3 litres of solution

a) an inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter paper to give a final concentration of 10 mg carbon/L
c) The test item on a filter paper, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item on a filter paper, in duplicate, in inoculated mineral medium plus an aliquot (20 mL) of a 10 g/L aqueous solution of sodium azide to give a final concentration of 10 mg carbon/L.

A filter paper was added to the inoculum control and procedure control 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 21±1°C in darkness.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

65

Sampling time:

28 d

Details on results:

The biotic test item attained 65% after 28 days and 70% degradation after 29 days. The abiotic test item attained 0% degradation after 28 days.

Chemical analysis of the biotic test preparations at Day 29 showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 5.3 mg/l/ The abiotic test sample had a concentration of 48% of nominal.

Analysis of the test preparations of the abiotic test vessels at Day 29 showed the losses from the test system to be via several mechanisms, not solely via biological pathways. Losses from the system were considered to be mainly via volatisation due to the chemical nature of many of the constituents of the test item but may also be due to losses due to formation of non-extractable residues.

Results with reference substance:

Sodium benzoate attained 70% degradation after 14 days and 81% degradation after 28 days.

Table 1: Percentage biodegradation values

Day	% Biodegradation
	Sodium benzoate Procedure control	Test item	Abiotic test item
0	0	0	0
2	41	3	0
6	53	4	0
8	54	8	0
10	61	17	0
14	70	40	0
21	79	58	0
28	81	65	0
29*	79	70	0

*Day 29 value was corrected to include any carry over of CO₂detected in Absorber 2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 65% degradation in 28 days (CO2 evolution) in a study conducted in accordance with OECD 301B.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-08-09 to 2013-11-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance (covering the carbon range from C18 to C30) with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test System
A mixed population of sewage treatment micro-organisms was obtained on 03 September 2013 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21±1ºC prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Experimental Preparation
Test Item
For the purpose of the test, following a procedure previously used by the sponsor on a similar test items, the test item was adsorbed onto glass-fibre filter paper (Whatman GF/A, 21 mm diameter) prior to addition to the test vessel.

An amount of test item (50 mg) was adsorbed onto glass-fibre filter paper supported on aluminium foil prior to addition into the test vessel. Mineral medium (495 mL) and inoculum (5 mL) was added to the test vessel to give a test concentration of 100 mg/L.

A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline.

Inoculum control vessels were prepared containing mineral medium (495 mL) and inoculum (5 mL).

Reference Item
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.

A nominal amount of test item (50 mg) was weighed onto glass-fibre filter paper supported by aluminium foil prior to addition into the test vessel. Mineral medium (445 mL) and an aliquot (50 mL) of the 1000 mg/L aniline stock solution were added to the test vessel prior to the addition of inoculum (5 mL) to give the test concentration of 100 mg test item/L and 100 mg aniline/L.


Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Five replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L.
c) Five replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing inoculated mineral medium the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L to act as toxicity control vessels.
e) Four replicates bottles containing the test item at a concentration of 100 mg/L and sodium azide at a concentration of 200 mg/L to act as abiotic test vessels.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.

A glass-fibre filter paper (Whatman GF/A, 21 mm diameter) supported on aluminium foil was added to each control and reference item vessel to maintain consistency between these vessels and the test item vessels.

Data from the inoculum control and procedure control vessels were shared with similar concurrent studies.

In order to confirm that the aniline stock solution was prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for DOC analysis.

Two of the five inoculum control, abiotic test vessels and test item vessels were sacrificed for immediate chemical analysis. All remaining inoculum control, abiotic test vessels and test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.

The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.

The test was conducted in diffuse light at a temperature of approximately 21±1ºC.

An additional replicate vessel was prepared for each treatment and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test. On Day 28 an assessment of the biological oxygen demand data was made and the most consistent vessels chosen for calculating and reporting purposes. The remaining vessels were discarded and are not reported.

Evaluations
Oxygen consumption measurements:
The daily Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and toxicity control are reported in the table below.

Physico-chemical measurements:
The temperatures of the water bath was recorded daily.

pH measurements:
On Day 0, the pH of each of the inoculum control and procedure control vessels was determined prior to the addition of the inoculum using a WTW pH/Oxi 340I pH and dissolved oxygen meter. The pH values were adjusted where necessary to pH 7.4±0.2 using dilute hydrochloric acid. The required quantity of inoculum was then added to each vessel.

On Day 28, the pH of the inoculum control and procedure control vessels was determined.

Due to the oily nature of the test item, it was considered inappropriate to determine the pH of the test item and toxicity control vessels on Day 0 and Day 28 as insertion of a pH probe into the test preparations may have resulted in the loss of test item by adherence to the pH probe.

Compound specific analysis:
on Day 0, two inoculum control and two test item vessels were sacrificed for compound specific analysis. On Day 28, chemical analysis of the two two inoculum control and test item vessels from which the oxygen consumption values were taken was performed.

Data evaluation

Calculation of Theoretical Oxygen Demand:
The Theoretical Oxygen Demand (ThOD) for a compound CcHhClclNnPpSsOoNana was calculated by:

ThOD (NO3)(mgO2 / mg) = (16(2c + ½(h-lc) + 4/2n + 5/2p + 3s + ½na – o) / molecular weight


Percentage Biodegradation:
The percentage biodegradation in terms of oxygen consumption was calculated as follows:

% degradation = (BOD B / ThOD) x 100

Where:
BOD = Biological Oxygen Demand of the test item or reference item (mgO2/L)
B = Oxygen consumption in basal mineral medium to which inoculum is added (control) (mgO2/L)
ThOD = Theoretical oxygen demand to completely oxidize the reference and/or test item (mgO2/L)

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

51

Sampling time:

28 d

Details on results:

Biodegradation
The test item attained 51% biodegradation after 28 days based on oxygen consumption.

The toxicity control attained 40% biodegradation after 14 days and 59% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:

Aniline (procedure control) attained 76% biodegradation after 14 days and 92% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Chemical analysis:

In an initial test that was terminated due to excessive BOD values in the control and test vessels, the results of the Day 0 analyses showed consistent results. However, in the repeat (definitive) test, the Day 0 analyses showed variable results with measured concentrations of 95% and 54% of nominal values being obtained. Since the initial test preparation yielded consistent results, it was decided to continue with the test but to also re-prepare another set of Day 0 samples for analysis to assess the variability. The third set of Day 0 samples that were prepared again showed low and variable results, 55% and 49% nominal .

Discussions with the sponsor suggested that the use of preliminary absorption onto filter paper may have contributed to the variation in Day 0 analyses. Therefore, a fourth set of Day 0 samples in which the test item was added directly to the test vessels were prepared. The results of these analyses showed consistent, near nominal results were obtained.

It was therefore considered that the low and variable results obtained from Day 0 analysis of the test vessels from the definitive test were a result of incomplete recovery of the test item after adsorption onto filter paper rather than incorrect preparation. It was therefore considered that the incubated test vessels contained the required concentration of the test item.

Analysis of the Day 28 test sample showed a decline in measured concentrations to 32% and 35% of nominal values, equivalent to 68% and 65% loss of test item over the 28 day test period.

Validity criteria fulfilled:

yes

Conclusions:

GTL Base Oil 3 has been tested in an OECD 301F (manometric respirometry) test conducted in compliance with GLP. The test substance attained 51% biodegradation in 28 days. Based on the results of the chemical analyses conducted, a 67% loss of test material occurred over the 28 day test period.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 21 December 2005 and 18 January 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance (covering the carbon range from C18 to C30) with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: ISO Guideline No. 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)"

Deviations:

yes

Remarks:

method adapted to provide a screening test at the request of the Sponsor.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.

- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air* for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until use.
(*CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb) granules)

- 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.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

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 21 December 2005 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage

TEST SYSTEM

The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:
a) A control consisting of inoculated culture medium, plus 100 mg silica gel 11 replica vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 20 mg carbon/l, 11 replica vessels.
c) The test material in inoculated culture, plus 100 silica gel, to give a final concentration of 20 mg carbon/l, 7 replica vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as toxicity control, 7 replicate vessels.

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.

Test media a-d were inoculated with the prepared inoculum at final concentration of 100 ml/l.

Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analysis on days 0 and 28 (duplicate vessels per sampling occasion).

All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20 ± 1 °C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).

Data from the control and standard material vessels was shared with similar concurrent studies.

DATA EVALUATION
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material was determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:
Carbon: 85.60% w/w
Hydrogen: 14.98% w/w
Nitrogen: 0.05% w/w

Thus for a test concentration of 20 mg C/l (a total of 2.50 mg) the total organic carbon present was 2.14 mg C.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6H5COONa) was calculated as follows:

[(No. of C atoms x mol wt of C)/mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11] x 100 = 58.34%


CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l


Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This must be reached within 10 days of the biodegradation exceeding 10%.

The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.

The toxicity control should attain ≥ 25% def=gradation by Day 14 for the test material to be considered as non-inhibitory.

The TIC produced from the control bottles at the end of the test should be ≤ 15% of the TOC added initially as test material.

Reference substance:

other: Sodium benzoate

Preliminary study:

Preliminary work conducted showed that a volume of 3.1 µl of test material measured using a gas tight micro-syringe (SGE P/N 001100 5FX) gave a measured weight of 2.50 mg (SD = 0.022 mg), mean of 15 separate weighings.

Test performance:

At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.

Parameter:

% degradation (CO2 evolution)

Value:

65

Sampling time:

28 d

Details on results:

The mean TIC in the control vessels on Day 28 was 0.18 mg/l; equivalent to 2% of the organic carbon added initially as test material to the vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards (ISO 1996) and in the published literature (Handley et al, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the medium and to increase the surface area of the test material exposed to the test organisms.

The test material attained 65% degradation after 28 days.

The toxicity control attained 73% degradation after 14 days and 76% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Results with reference substance:

Sodium benzoate attained 65% degradation after 14 days and 85% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R 2. The degradation rates for the standard material were similar to those determined by IC analyses.

3 cSt Fluid: Assessment of Ready Biodegradability; CO2 In Sealed Vessels (CO2 Headspace Test)

Percentage Biodegradation value:

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
14	65	52	73
28	84	65	76

Validity criteria fulfilled:

yes

Interpretation of results:

other: Result cannot be interpreted to indicate that the sample was readily biodegradable, though it does suggest significant intrinsic biodegradability.

Conclusions:

At the end of the 28 d test period, 65% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes. This result cannot be interpreted to indicate that the sample was readily biodegradable, though it does suggest significant intrinsic biodegradability.

Executive summary:

An ISO 14593 study with 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (3 cSt) has been conducted. Note: Elemental analysis was conducted, which revealed a small proportion of nitrogen present in the test substance (0.05%). The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking. A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance. Assessment of biodegradation was made by CO₂analysis only, based on 2-weekly samplings.

At the end of the 28 d test period, 65% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes. This result cannot be interpreted to indicate that the sample was readily biodegradable, though it does suggest significant intrinsic biodegradability.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 November 2006 - 7 December 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance (covering the carbon range from C18 to C30) with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds ill aqueous medium - Method by analysis of inorganic carbon in sealed vessels (C02 headspace test)"

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.

- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately I hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the
supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until 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.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SPECIES:
A mixed population of sewage sludge micro-organisms was obtained on 9 November 2006 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

TEST SYSTEM

The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:

a) A control consisting of inoculated culture medium, plus 100 mg silica gel, 11 replicate vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of20 mg carbon/l, 11 replicate vessels.
c) The test material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of mg carbon/I, 7 replicate vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 7 replicate vessels.

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.

Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 mI/l.

Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).

All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20± 1°C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).

Data from the control and standard material vessels was shared with similar concurrent studies.

SAMPLING
The slight variation in the degradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the nature of the study design (individual replicates sacrificed on each sampling occasion), the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected.


Dissolved Organic Carbon (DOC) analyses
Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R2. The degradation rates for the standard material were similar to those determined by IC analyses.


CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/I, equivalent to a total of20 mg carbon/I.

EVALUATION OF DATA
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material was determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:

Carbon: 85.44% w/w
Hydrogen: 15.01% w/w
Nitrogen: 0.09% w/w

Thus for a test concentration of 20 mg C/I (a total of2.50 mg) the total organic carbon present was 2.14mgC.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6H6COONa) was calculated as follows:

[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%

Thus for a 20 mg C/I test concentration (34.3 mg/I) the total organic carbon present in each test vessel was 2.14mgC.

Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.

The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.

The toxicity control should attain ≥ 25% degradation by Day 14 for the test material to be considered as noninhibitory.

The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test material.

Reference substance:

other: Sodium benzoate

Preliminary study:

Preliminary work conducted showed that a volume of 3.2 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE PIN 001 100 5FX) gave a measured weight of 2.51 mg, mean of 15 separate weighings.

Test performance:

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Parameter:

% degradation (CO2 evolution)

Value:

43

Sampling time:

28 d

Details on results:

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards Organisation (ISO 1996) and in the published lilerature (Handley et ai, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The test material attained 43% degradation after 28 days.

The toxicity control attained 60% degradation after 14 days and 66% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that
the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.

Results with reference substance:

Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable.

Conclusions:

The test material attained 43% degradation after 28 days.The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (3 cSt) is not readily biodegradable.

Executive summary:

An ISO 14593 study with 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (3 cSt) has been conducted. The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking. A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance. Assessment of biodegradation was made by CO₂analysis only, based on 2-weekly samplings. At the end of the 28 d test period, 43% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes. The study demonstrates that the substance is not readily biodegradable.

Conclusion: The test material attained 43% degradation after 28 days. The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (3 cSt) is not readily biodegradable.

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 November 2006 - 7 December 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance (covering the carbon range from C21 to C50) with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds ill aqueous medium - Method by analysis of inorganic carbon in sealed vessels (C02 headspace test)"

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.

- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately I hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the
supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until 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.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SPECIES:
A mixed population of sewage sludge micro-organisms was obtained on 9 November 2006 from the secondary treatment stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

TEST SYSTEM

The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:

a) A control consisting of inoculated culture medium, plus 100 mg silica gel, 11 replicate vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of20 mg carbon/l, 11 replicate vessels.
c) The test material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of mg carbon/I, 7 replicate vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 7 replicate vessels.

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.

Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 mI/l.

Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).

All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20± 1°C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).

Data from the control and standard material vessels was shared with similar concurrent studies.

SAMPLING
The slight variation in the degradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the nature of the study design (individual replicates sacrificed on each sampling occasion), the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected.


Dissolved Organic Carbon (DOC) analyses
Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R2. The degradation rates for the standard material were similar to those determined by IC analyses.


CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/I, equivalent to a total of20 mg carbon/I.

EVALUATION OF DATA
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material were determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:

Carbon: 85.44% w/w
Hydrogen: 15.01% w/w
Nitrogen: 0.09% w/w

Thus for a test concentration of 20 mg C/I (a total of2.50 mg) the total organic carbon present was 2.14mgC.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6H,COONa) was calculated as follows:

[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%

Thus for a 20 mg C/I test concentration (34.3 mg/I) the total organic carbon present in each test vessel was 2.14mgC.

Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.

The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.

The toxicity control should attain ≥ 25% degradation by Day 14 for the test material to be considered as noninhibitory.

The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test material.

Reference substance:

other: Sodium benzoate

Preliminary study:

Preliminary work conducted showed that a volume of 3.2 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE PIN 001 100 5FX) gave a measured weight of 2.51 mg, mean of 15 separate weighings.

Test performance:

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Parameter:

% degradation (CO2 evolution)

Value:

24

Sampling time:

28 d

Details on results:

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% of the organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The test material attained 24% degradation after 28 days.

The toxicity control attained 56% degradation after 14 days and 58% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that
the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.

Results with reference substance:

Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable.

Conclusions:

The test material attained 24% degradation after 28 days. The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) is not readily biodegradable.

Executive summary:

An ISO 14593 study with the substance 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) has been conducted. The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking. A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance. Assessment of biodegradation was made by CO₂analysis only, based on 2-weekly samplings. At the end of the 28 d test period, 24% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes. The study demonstrates that the substance is not readily biodegradable.

Conclusion: The test material attained 24% degradation after 28 days. The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) is not readily biodegradable.

Reference 7

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 November 2006 - 7 December 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance (covering the carbon range from C21 to C50) with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds ill aqueous medium - Method by analysis of inorganic carbon in sealed vessels (C02 headspace test)"

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.

- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately I hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the
supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until 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.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SPECIES:
A mixed population of sewage sludge micro-organisms was obtained on 9 November 2006 from the secondary treatment stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

TEST SYSTEM

The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:

a) A control consisting of inoculated culture medium, plus 100 mg silica gel, 11 replicate vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of20 mg carbon/l, 11 replicate vessels.
c) The test material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of mg carbon/I, 7 replicate vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 7 replicate vessels.

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.

Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 mI/l.

Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).

All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20± 1°C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).

Data from the control and standard material vessels was shared with similar concurrent studies.

SAMPLING
The slight variation in the degradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the nature of the study design (individual replicates sacrificed on each sampling occasion), the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected.


Dissolved Organic Carbon (DOC) analyses
Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R2. The degradation rates for the standard material were similar to those determined by IC analyses.


CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/I, equivalent to a total of20 mg carbon/I.

EVALUATION OF DATA
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material was determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:

Carbon: 85.44% w/w
Hydrogen: 15.01% w/w
Nitrogen: 0.09% w/w

Thus for a test concentration of 20 mg C/I (a total of2.50 mg) the total organic carbon present was 2.14mgC.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6H,COONa) was calculated as follows:

[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%

Thus for a 20 mg C/I test concentration (34.3 mg/I) the total organic carbon present in each test vessel was 2.14mgC.

Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.

The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.

The toxicity control should attain ≥ 25% degradation by Day 14 for the test material to be considered as noninhibitory.

The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test material.

Reference substance:

other: Sodium benzoate

Preliminary study:

Preliminary work conducted showed that a volume of 3.2 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE PIN 001 100 5FX) gave a measured weight of 2.51 mg, mean of 15 separate weighings.

Test performance:

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Parameter:

% degradation (CO2 evolution)

Value:

29

Sampling time:

28 d

Details on results:

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards Organisation (ISO 1996) and in the published lilerature (Handley et ai, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The test material attained 29% degradation after 28 days.

The toxicity control attained 49% degradation after 14 days and 57% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that
the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.

Results with reference substance:

Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable.

Conclusions:

The test material attained 29% degradation after 28 days. The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) is not readily biodegradable.

Executive summary:

An ISO 14593 study with the substance 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) has been conducted. The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking. A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance. Assessment of biodegradation was made by CO₂analysis only, based on 2-weekly samplings. At the end of the 28 d test period, 29% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes. The study demonstrates that the substance is not readily biodegradable.

Conclusion: The test material attained 29% degradation after 28 days. The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) is not readily biodegradable.

Reference 8

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The definitive study was conducted between 20 January 2004 and 18 February 2004 and the last date that data was acquired for the study was 19 February 2004.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C21 to C50

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 2nd December 2002 Date of signature: 13 February 2003

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- 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 maintained on continuous aeration in the laboratory at a
temperature of 21°C and was 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 activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. The filter paper was then dried in an oven at approximately 105°C for at least I hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
The suspended solids were equal to 3.3 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.

Duration of test (contact time):

28 d

Initial conc.:

10.1 mg/L

Based on:

test mat.

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 19 January 2004 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage

TEST SYSTEM

The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 Iitres of solution:

a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel.
b) The standard material (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 material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10.1 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20.1 mg carbon/l to act as a toxicity control (one vessel only).

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/1. The study was carried out in a temperature controlled room at 21 °C, in darkness.

Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 27.3 ml of inoculum and aerated overnight. On Day 0 the test and standard materials 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
-CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.

The samples taken on Days 0 from the first absorber vessels and the samples taken on Days 1, 2, 3, 6, 8, 10, 14, 16, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Day 0 from the second absorber vessels were stored at approximately -20 °C prior to analysis. The samples taken on Days 12 and 18 were also stored at approximately 20 °C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed degradation did not attain 60% and therefore additional analyses were considered to be unnecessary.

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 an Ionies 1555B TOC analyser. Samples (300 or 40 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 by orthophosphoric acid using zero grade air or nitrogen (oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2C03). Each analysis was carried out in triplicate.

Dissolved Organic carbon (DOC) analysis:
Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material 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 material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material 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-VCSH TOC analyser. Samples (27 or 13 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 Measurement:

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter and dissolved oxygen meter.


CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (35.4 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 100 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 10 minutes. The test material/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 11.8 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20.1 mg carbon/l.


The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total Co2 evolution = mgIC in control x (100/%C of Co2) x (1/test volume).

EVALUATION OF DATA
Calculation of carbon content
The test material contains 85.50% carbon and so for a concentration of 10.1 mg C/I (a total of35.4 mg) the total organic carbon present was 30.3 mg C.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6HSCOONa) was calculated as follows:

[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%

Thus for a 10 mg C/I test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.


Validation criteria:
The results of the degradation test are considered valid if in the same test the standard material yields ~ 60% degradation by Day 14.

The test material 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 material and sodium benzoate) should attain ≥ 25% degradation by Day 14 for the test material 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 material suspension in the mineral medium at the beginning of the test should be < 5% of the TC.

Reference substance:

other: Sodium benzoate

Preliminary study:

At the request of the Sponsor and in view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organization (ISO 1996) and in the published literature (Handley et ai, 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels.
Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.

Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

Parameter:

% degradation (CO2 evolution)

Value:

46

Sampling time:

28 d

Details on results:

The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test 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.

At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.

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 standard material Replicate R2. These increases were considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in an increase in the percentage degradation value for the test material from 44% on Day 28 to 46% on Day 29.

The addition of the acid causes all biological activity to stop, and so the test material attained 46% degradation after 28 days as CO2 was present in solution on Day 28 but was not released until after the addition of the acid. The test material, therefore, cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301H.

Variation in degradation rates for the test material on different sampling days was considered to be due to variation in the CO2 production rates between the replicate control and replicate test vessels.

The toxicity control attained 58% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 47% on Day 28 to 58% on Day 29.

Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation values for the standard material from 69% on Day 28 to 71% on Day 29.

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.

Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC) gave percentage degradation values of 101% and 91% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analysis were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions of inoculum and silica gel with no undissolved standard material visible. The test material vessels were cloudy light brown dispersions of the test material, inoculum and silica gel. The toxicity control vessel was a cloudy light brown dispersion of test material, inoculum and silica gel with no undissolved standard material.

Results with reference substance:

Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the standard material from 69% o Day 28 to 71% on Day 29.

Percentage Biodegradation value:

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	22	4	1
2	29	2	0
3	43	1	0
6	50	7	35
8	54	10	35
10	58	13	37
14	62	23	37
16	63	28	50
20	67	36	53
22	66	35	53
24	67	41	56
27	69	45	50
28	69	44	47
29*	71	46	58

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

Validity criteria fulfilled:

yes

Interpretation of results:

other: Not Readily Biodegradable

Conclusions:

The test material attained 46% degradation after 28 days and therefore cannot 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 test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).’

Method: The test material, at a concentration of 10.1 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed vessels in the dark at a temperature of 21 °C for 28 days.

At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium i n order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

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

Results & Conclusion: The test material attained 46% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Reference 9

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The definitive study was conducted between 20 January 2004 and 18 February 2004 and the last date that data was acquired for the study was 19 February 2004.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C27 to C57

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 2nd December 2002 Date of signature: 13 February 2003

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- 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 maintained on continuous aeration in the laboratory at a
temperature of 21°C and was 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 activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. The filter paper was then dried in an oven at approximately 105°C for at least I hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
The suspended solids were equal to 3.3 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.

Duration of test (contact time):

28 d

Initial conc.:

10.1 mg/L

Based on:

test mat.

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 19 January 2004 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage

TEST SYSTEM

The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 Iitres of solution:

a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel.
b) The standard material (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 material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10.1 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20.1 mg carbon/l to act as a toxicity control (one vessel only).

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/1. The study was carried out in a temperature controlled room at 21 °C, in darkness.

Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 27.3 ml of inoculum and aerated overnight. On Day 0 the test and standard materials 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
-CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.

The samples taken on Days 0 from the first absorber vessels and the samples taken on Days 1, 2, 3, 6, 8, 10, 14, 16, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Day 0 from the second absorber vessels were stored at approximately -20 °C prior to analysis. The samples taken on Days 12 and 18 were also stored at approximately 20 °C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed degradation did not attain 60% and therefore additional analyses were considered to be unnecessary.

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 an Ionies 1555B TOC analyser. Samples (300 or 40 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 by orthophosphoric acid using zero grade air or nitrogen (oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2C03). Each analysis was carried out in triplicate.

Dissolved Organic carbon (DOC) analysis:
Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material 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 material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material 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-VCSH TOC analyser. Samples (27 or 13 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 Measurement:

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter and dissolved oxygen meter.


CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (35.4 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 100 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 10 minutes. The test material/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 11.8 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20.1 mg carbon/l.


The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total Co2 evolution = mgIC in control x (100/%C of Co2) x (1/test volume).

EVALUATION OF DATA
Calculation of carbon content
The test material contains 85.50% carbon and so for a concentration of 10.1 mg C/I (a total of 35.4 mg) the total organic carbon present was 30.3 mg C.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6HSCOONa) was calculated as follows:

[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%

Thus for a 10 mg C/I test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.


Validation criteria:
The results of the degradation test are considered valid if in the same test the standard material yields ≥ 60% degradation by Day 14.

The test material 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 material 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 material suspension in the mineral medium at the beginning of the test should be < 5% of the TC.

Reference substance:

other: Sodium benzoate

Preliminary study:

At the request of the Sponsor and in view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels.
Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC/TC ratio of the test material suspension in the mineral medium at the start of the test was below 5% and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for C02 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.

Parameter:

% degradation (CO2 evolution)

Value:

55

Sampling time:

28 d

Details on results:

The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test 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.

At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.

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 standard material Replicate R2. These increases were considered to be due to C02 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in an increase in the percentage degradation value for the test material from 48% on Day 28 to 55% on Day 29.

The addition of the acid causes all biological activity to stop, and so the test material attained 55% degradation after 28 days as CO2 was present in solution on Day 28.

The increase in inorganic carbon in the test material Replicate R2 first absorber vessel resulted in an increase in the percentage degradation value for the test material from 10% on Day 28 to 11% on Day 29.

Variation in degradation rates for the test material on different sampling days was considered to be due to variation in the CO2 production rates between the replicate control and replicate test vessels.

The toxicity control attained 55% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 47% on Day 28 to 55% on Day 29.

Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the standard material from 69% on Day 28 to 71% on Day 29.

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.

Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC) gave percentage degradation values of 101% and 91% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analysis were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions of inoculum and silica gel with no undissolved standard material visible. The test material vessels were cloudy light brown dispersions of the test material, inoculum and silica gel. The toxicity control vessel was a cloudy light brown dispersion of test material, inoculum and silica gel with no undissolved standard material.

Results with reference substance:

Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the standard material from 69% on Day 28 to 71% on Day 29.

Percentage Biodegradation value:

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	22	2	9
2	29	2	14
3	43	0	27
6	50	0	33
8	54	2	34
10	58	0	35
14	62	13	41
16	63	13	41
20	67	9	43
22	66	5	48
24	67	12	48
27	69	16	49
28	69	10	47
29*	71	11	55

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

Validity criteria fulfilled:

yes

Interpretation of results:

other: not Readily Biodegradable

Conclusions:

The test material attained 11% degradation after 28 days and therefore cannot 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 test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ (8 cSt) in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m)).

Method: The test material, at a concentration of 10.1 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed vessels in the dark at a temperature of 21 °C for 28 days.

At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium i n order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

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

Results & Conclusion: The test material attained 11% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Reference 10

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August 2013 to 12 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3100 (Aerobic Aquatic Biodegradation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

Test System
A mixed population of sewage treatment micro-organisms was obtained on 03 September 2013 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Procedure
Experimental Preparation
Test Item
For the purpose of the test, following a procedure previously used by the Sponsor on similar test items, the test item was adsorbed onto glass-fibre filter paper (Whatman GF/A, 21 mm diameter) prior to addition to the test vessels.

An amount of test item (50 mg) was adsorbed onto glass-fibre filter paper supported on aluminium foil prior to addition into the test vessel. Mineral medium (495 mL) and inoculum (5 mL) was added to the test vessel to give the test concentration of 100 mg/L. .

A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline.

Inoculum control vessels were prepared containing mineral medium (495 mL) and inoculum (5 mL).

Reference Item
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.


Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.

An amount of test item (50 mg) was adsorbed onto glass-fibre filter paper supported on aluminium foil prior to addition into the test vessel. Mineral medium (445 mL) and an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.2.2) were added to the test vessel prior to the addition of inoculum (5 mL) to give the test concentration of 100 mg test item/L and 100 mg aniline/L.


Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:

a) Five replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L.
c) Five replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing inoculated mineral medium the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L to act as toxicity control vessels.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.

A glass-fibre filter paper (Whatman GF/A, 21 mm diameter) supported on aluminium foil was added to each control and reference item vessel to maintain consistency between these vessels and the test item vessels.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

In order to confirm that the aniline stock solution was prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for DOC analysis.

Two of the five inoculum control and test item vessels were sacrificed for immediate chemical analysis (see Appendix 2). All remaining inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.

The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.

The test was conducted in diffuse light at a temperature of 21 ± 1 ºC.

An additional replicate vessel was prepared for each treatment and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test. On Day 28 an assessment of the biological oxygen demand data was made and the most consistent vessels chosen for calculating and reporting purposes. The remaining vessels were discarded and are not reported.

Evaluations
Oxygen Consumption Measurements
The daily Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control are given Table 1.


Physico-chemical Measurements
The temperature of the water bath was recorded daily.


pH Measurements
On Day 0 the pH of each of the inoculum control and procedure control vessels was determined prior to the addition of the inoculum using a WTW pH/Oxi 340I pH and dissolved oxygen meter. The pH values were adjusted where necessary to pH 7.4 ± 0.2 using diluted hydrochloric acid. The required quantity of inoculum was then added to each vessel.

On Day 28 the pH of the inoculum control and procedure control vessels was determined.

Due to the oily nature of the test item it was considered inappropriate to determine the pH of the test item and toxicity control vessels on Days 0 and 28 as insertion of a pH probe into the test preparations may have resulted in the loss of test item by adherence to the pH probe.

Compound Specific Analyses
On Day 0, two inoculum control and two test item vessels were sacrificed for compound specific analysis. On Day 28 chemical analysis of the two inoculum control and test item vessels from which the oxygen consumption values were taken was performed (see Appendix 2).

Reference substance:

aniline

Preliminary study:

None

Test performance:

None

Parameter:

% degradation (O2 consumption)

Value:

68

Sampling time:

28 d

Details on results:

Results
Daily BOD values for the test item, procedure control, toxicity control and inoculum control vessels are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2. BOD curves and biodegradation curves are given in Figures 2 and 3 respectively. The pH values of each individual vessel on Days 0 and 28 are given in Table 3.

The calculated Theoretical Oxygen Demand values for the test and procedure control are given in Appendix 4.


Validation Criteria
The mean BOD of the inoculated mineral medium (control) was 65.93 mg O2/L after 28 days. This was slightly higher than the upper limit of 60 mg O2/L recommended in the Test Guideline, however given that biodegradation of the reference item satisfied the validation criteria and that there were no abnormal observations throughout the test period, the slightly higher control BOD was considered not to affect the integrity of the test.

The difference between extremes of replicate BOD values at the end of the test and at the end of the 10-Day window was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.


Biodegradation
The test item attained 68% biodegradation after 28 days and can therefore be considered to be readily biodegradable. The test item failed to meet the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, Paragraph 43 (Page 8), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10-Day window should not be applied to interpret the results of the test.

The toxicity control attained 67% biodegradation after 14 days and 87% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.


Chemical Analysis
In an initial test that was terminated due to excessive BOD values in the control and test vessels, the results of the Day 0 analyses showed near nominal measured concentrations (80% and 90%, see Appendix 2). However in the repeat (definitive) test the Day 0 analyses showed lower results with measured concentrations of 69% and 70% of nominal values being obtained. Since the initial test preparation yielded near nominal results it was decided to continue with the test but to also re-prepare another set of Day 0 samples for analysis to assess the variability. The third set of Day 0 samples that were prepared again showed low and variable results, 65% and 52% of nominal.

Discussions with the Sponsor suggested that the use of preliminary absorption onto filter paper may have contributed to the variation in Day 0 analyses. Therefore a fourth set of Day 0 samples in which the test item was added directly to the test vessels were prepared. The results of these analyses showed consistent, near nominal results were obtained (see Appendix 2).

It was therefore considered that the low results obtained from the Day 0 analysis of the test vessels from the definitive test were a result of incomplete recovery of the test item after adsorption onto filter paper rather than incorrect preparation. It was therefore considered that the incubated test vessels contained the required concentration of test item.

Analysis of the Day 28 test samples showed a decline in measured concentrations to 5% and 8% of nominal values, equivalent to 92% and 95% loss of test item over the 28-Day test period.

DISCUSSION
In a separate study conducted on the test item in which abiotic loss of the test item from the test system was assessed (Harlan Study Number 41302225), the test item concentration declined by approximately 40% over a 28-Day period. This loss was attributed to the volatility of the test item resulting in some partitioning between the aqueous phase and the headspace. Then as the test vessel was opened to sample for analysis at Day 28, losses of the test item from the headspace into the atmosphere occurred.

In this study to assess the biodegradation of the test item over a 28-Day period in an inoculated test system, the losses of test item observed by chemical analysis were higher than those that could be attributed to abiotic losses alone. Combined with the oxygen consumption data generated it is clear that significant biological degradation of the test item occurs in an inoculated test system.

Another factor in the apparent reduced biodegradation value based on oxygen consumption values compared to the losses calculated from the chemical analyses conducted may be due to incorporation of the test item, or degradation products of the test item, into the microbial biomass. In such cases the micro-organisms present utilize carbon originating from the test item to increase their biomass by incorporating the carbon into new cells. This effectively removes the test item from the aqueous phase and hence reduces the apparent biodegradation of the test item as measured by oxygen consumption.


It should also be noted that all the analysis of the Day 0 vessels indicated that the concentrations achieved were generally lower than the nominal concentrations on which the theoretical oxygen demand were based. This could have led to an underestimation of the biodegradation calculated on the basis of oxygen consumption.

In the ECHA R.7b Guidance (ECHA, 2012, page 193) it states that
“Degradation can be monitored by either measuring the complete breakdown of the chemical to carbon dioxide and water (ultimate degradation), or simply the measuring the disappearance of the parent substance, primary degradation. While ultimate degradation is preferred, primary degradation can be used to define the pass levels in each of the degradation tests provided certain conditions are met. Data on primary biodegradability may be used for demonstrating rapid degradability only when it can be satisfactorily demonstrated that the degradation products formed do not fulfil the criteria for classification as hazardous to the aquatic environment.”

The test item is comprised of alkanes and iso-alkanes which would not be anticipated to breakdown into hazardous components. Therefore, based on loss of parent substance the test substance would also be considered to be readily biodegradable.

CONCLUSION
The test item attained 68% biodegradation after 28 days based on oxygen consumption values.

Based on the results of the chemical analyses conducted, 94% removal of test item occurred over a 28-Day period.

The test item can therefore be considered to be readily biodegradable on the basis of both parent substance removal and oxygen consumption.

Results with reference substance:

Aniline (procedure control) attained 76% biodegradation after 14 days and 93% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions

Tables

Table1     Biological Oxygen Demand (BOD) Values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test Item		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	0.08	0.54	0.58	0.12	0.16	0.88
2	3.24	4.16	3.70	6.84	4.00	4.66
3	5.78	7.34	8.16	22.70	20.66	11.12
4	8.04	10.08	11.70	44.10	42.20	42.40
5	10.32	12.96	35.36	56.76	54.32	133.08
6	13.12	16.16	126.16	72.76	70.84	220.20
7	15.42	18.74	199.54	98.80	96.08	287.72
8	17.74	21.54	217.96	126.82	118.78	319.66
9	19.78	24.08	229.20	144.40	135.86	349.32
10	21.62	26.44	237.74	158.86	150.74	378.30
11	23.40	28.78	245.56	172.44	164.14	407.46
12	25.00	30.82	253.32	185.64	175.92	429.36
13	26.82	33.16	260.52	198.04	187.30	447.44
14	28.32	35.16	265.76	208.80	197.84	463.44
15	30.32	37.60	273.30	218.92	208.00	479.56
16	32.16	39.78	280.56	229.04	217.08	495.46
17	34.36	42.44	288.26	239.90	225.78	513.08
18	36.36	44.90	290.68	249.86	233.32	529.92
19	38.52	47.48	292.64	258.86	240.44	544.50
20	40.82	50.14	297.34	266.90	247.16	557.82
21	43.32	52.82	297.34	273.94	253.20	570.40
22	46.06	55.26	297.34	279.72	258.64	582.48
23	48.44	57.56	302.12	284.42	263.52	593.52
24	50.82	60.18	312.42	288.68	268.10	602.92
25	53.18	63.10	324.42	292.88	272.56	610.80
26	55.52	66.10	333.58	297.18	277.06	617.66
27	57.72	68.88	342.90	301.84	281.56	623.92
28	60.02	71.84	352.70	306.00	285.72	630.00

 

Table2     Biodegradation Values

Day	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	0	1	0	1	0
3	1	5	4	5	1
4	1	10	10	10	5
5	8	13	13	13	19
6	36	17	17	17	32
7	59	24	23	24	42
8	64	32	29	31	46
9	67	36	34	35	50
10	69	40	37	39	55
11	71	43	41	42	59
12	73	46	44	45	62
13	75	49	46	48	64
14	76	52	49	51	67
15	77	54	51	53	69
16	79	57	53	55	71
17	81	59	55	57	73
18	81	62	57	60	75
19	81	63	58	61	77
20	82	65	59	62	79
21	81	66	60	63	80
22	80	67	61	64	82
23	81	68	62	65	83
24	83	69	63	66	84
25	86	69	63	66	85
26	88	70	64	67	86
27	90	70	64	67	86
28	93	71	65	68	87

 

Table3     pH Values of the Test Preparationson Days 0 and 28

 

Test Vessel	pH
	Day 0	Day 28
Inoculum ControlR1	7.5	7.8
Inoculum Control R2	7.6	7.9
Procedure Control	7.6	7.5

 

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 68% biodegradation after 28 days (on the basis of oxygen consumption) and can therefore be considered to be readily biodegradable. The test item failed to meet the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, Paragraph 43 (Page 8), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10-Day window should not be applied to interpret the results of the test.
Based on chemical analysis, 94% removal of test item was observed over the test period.

Executive summary:

The key ready biodegradation study is a recently conducted test performed with Distillates (Fischer-Tropsch), C8-26 – branched and linear, in accordance with OECD 301F (manometric respirometry) and in compliance with GLP. The substance was introduced into test flasks by first adsorbing 50 mg onto glass-fibre filter paper supported on aluminium foil prior to addition into the test vessel. The test substance concentration was 100 mg/l. A glass-fibre filter paper supported on aluminium foil was also added to each control and reference item vessel to maintain consistency between these vessels and the test item vessels. The system consisted of a sample flask sealed by a sensor head/CO₂trap immersed in a temperature controlled water bath. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. Assessment of biodegradation was made by oxygen consumption and chemical analysis using gas chromatography.

 

The test item attained 68% biodegradation after 28 days (on the basis of oxygen consumption) and can therefore be considered to be readily biodegradable. The test item failed to meet the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, Paragraph 43 (Page 8), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10-Day window should not be applied to interpret the results of the test.

Based on chemical analysis, 94% removal of test item was observed over the test period.

Reference 11

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 17 July 2014 and 22 August 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C15 to C19

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 17 July 2014 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.

Duration of test (contact time):

28 d

Details on study design:

Experimental Preparation
Test Item
At the request of the Sponsor and following the recommendations of the International Standards Organisation (ISO, 1995) the test item was dispensed onto a filter paper* using a gas tight syringe. Using this method enables relatively small amounts of test item to be added accurately to the test vessels.

An amount of test item (66 µL, equivalent to 50 mg of test item determined by preliminary weighings) was dispensed onto filter paper* which was resting on a piece of foil and added immediately to mineral medium (495 mL) and inoculum (5 mL) to give the test concentration of 100 mg/L. Due to the volatile nature of the test item each vessel was placed immediately on the respirometer after the addition of the inoculum.

A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline.
Inoculum control vessels were prepared containing mineral medium (495 mL) and inoculum (5 mL).

A filter paper* resting on a piece of foil was added to each inoculum control vessel in order to maintain consistency between the test and inoculum control vessels.

Abiotic Test
A nominal amount of sodium azide (10.00 g) was dissolved in mineral medium and the volume adjusted to 500 mL to give a 20 g/L stock solution.

An amount of test item (66 µL, equivalent to 50 mg of test item determined by preliminary weighings) was dispensed onto a filter paper* which was resting on a piece of foil and added immediately mineral medium (445 mL) with an aliquot (50 mL) of the 20 g/L sodium azide stock solution to give the test concentration of 100 mg test item/L and 2000 mg sodium azide/L. Due to the volatile nature of the test item each vessel was immediately placed on the respirometer after the addition of the inoculum.


Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:

a) Five replicate bottles containing inoculated mineral medium to act as the inoculum control plus a filter paper*.
b) Two replicate bottles containing inoculated mineral medium plus a filter paper* and the reference item, aniline, at a concentration of 100 mg/L.
c) Five replicate bottles containing inoculated mineral medium and the test item on a filter paper* at a concentration of 100 mg/L.
d) Two replicate bottles containing inoculated mineral medium, the reference item, aniline, at a concentration of 100 mg/L and the test item on a filter paper* at a concentration of 100 mg/L to act as toxicity control vessels.
e) Four replicate bottles containing inoculated mineral medium, the test item on a filter paper* at a concentration of 100 mg/L and sodium azide at a concentration of 2000 mg/L to act as abiotic test vessels.

A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.

On Day 0 the reference item and sodium azide was added (where appropriate) and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter prior to the addition of test item (where appropriate). If necessary the pH values were adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the addition of the inoculum and test item.

Two of the five inoculum control and test item vessels and two of the four abiotic test vessels were sacrificed for immediate chemical analysis. All remaining inoculum control, test item, procedure control, toxicity control and abiotic test vessels were placed in a CES Multi-Channel Aerobic Respirometer.

The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.

The test was conducted in diffuse light at temperatures of approximately 24 ºC.

On Day 28 an assessment of the biological oxygen demand data was made and the most consistent vessels (two inoculum control, one procedure control, two test item, one toxicity control and two abiotic test vessels) chosen for calculating and reporting purposes. The remaining vessels were discarded and are not reported.

The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

Evaluations
Physico-chemical Measurements
The temperature of the water bath was recorded daily.


pH Measurements
On Day 0 the pH of each of the test vessels was determined prior to the addition of the inoculum and, where appropriate, the addition of the test item using a Hach HQ40d Flexi handheld meter. The pH values were adjusted where necessary to pH 7.4 ± 0.2 using diluted hydrochloric acid. The required quantity of inoculum and test item, where appropriate, was then added to each vessel.

On Day 28 the pH of the inoculum control and procedure control vessels was determined. Due to the volatile and oily nature of the test item it was considered inappropriate to determine the pH of the vessels containing test item.


Total Viable Counts
In order to confirm that abiotic conditions were present in the abiotic test vessels at the end of the test, total viable counts were performed. An aliquot (100 µL) of sample was dispensed onto a Tryptone Soya Agar (TSA) plate and spread over the plate prior to incubation at approximately 25 ºC for approximately 2 days. After the incubation period, the number of colony forming units (cfu) were determined by direct counting of the colonies on each agar plate.


Compound Specific Analyses
On Day 0, two inoculum control, two test item and two abiotic test vessels were sacrificed for compound specific analysis. On Day 28 chemical analysis of the two inoculum control, test item and abiotic test vessels from which the oxygen consumption values were taken was

Data Evaluation
Calculation of Theoretical Oxygen Demand
The Theoretical Oxygen Demand (ThOD) for a compound CcHhClclNnPpSsOoNana was calculated by:

ThOD (NO3)(mgO2/mg) = (16(2c + ½ (h-cl) + 5/2n +5/2p + 3s + ½ na – o) / molecular weight

Percentage Biodegradation
The percentage biodegradation in terms of oxygen consumption was calculated as follows:

% degradation = ((BOD-B) / ThOD) x 100

Where:
BOD = Biological Oxygen Demand of the test item or reference item (mgO2/L)
B = Oxygen consumption in basal mineral medium to which inoculum is added (control) (mgO2/L)
ThOD = Theoretical oxygen demand to completely oxidize the reference and/or test item (mgO2/L)

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

73

Sampling time:

28 d

Details on results:

Total Viable Counts
The total viable counts from the abiotic test vessels confirmed that abiotic conditions had been present as the number of total viable counts were very low.


Biodegradation
The test item attained 73% biodegradation after 28 days based on oxygen consumption values. The test item failed to meet the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals (OECD, 2006), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10-Day window should not be applied to interpret the results of the test.

The toxicity control attained 62% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Chemical Analysis
Chemical analysis of the 100 mg/L test preparation at 0 hours showed a mean measured concentration of 102% of nominal was obtained. A decline in the mean measured test concentration was observed on Day 28 to 7% of nominal (93% loss over the test duration assuming 100% recovery on Day 0).

The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to a combination of volatility of the test item and incorporation of the test item, or degradation products of the test item, into the microbial biomass. In such cases the micro-organisms present utilize carbon originating from the test item to increase their biomass by incorporation the carbon into new cells. This effectively removes the test item from the aqueous phase and hence reduces the apparent biodegradation of the test item as measured by oxygen consumption.

Chemical analysis of the abiotic test vessels on Day 0 showed a mean measured concentration of 108% of nominal was obtained. Analysis on Day 28 showed a mean measured concentration of 70% of nominal was obtained. Given the low total viable counts from the abiotic test vessels on Day 28 and the low biological oxygen consumption values from these vessels it was considered that this 35% loss of test item measured by chemical analysis on Day 28 occurred during sampling and analysis given the volatile nature of the test item.

Results with reference substance:

Aniline (procedure control) attained 69% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validation Criteria

The mean BOD of the inoculated mineral medium (control) was 24.36 mg O₂/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Theoretical Oxygen Demand Values

Calculation of Theoretical Oxygen Demand (ThOD) for the test and reference items.

Information supplied by the Sponsor indicated that the ThOD value for the test item was
3.40 mg O₂/mg.

Therefore for a test concentration of 100 mg/L, the ThOD will be 340 mg O₂/L.

 

Reference Item (Procedure Control): Aniline C₆H₅NH₂       mol wt = 93.13

 

ThOD (NO3) = ((16[12 + 3.5 + 2.5]) / 93.13) = 3.09 mg O2/mg

Therefore, for a test concentration of 100 mg/L, the ThOD will be 309 mg O₂/L.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In an OECD 301F manometric respirometry test conducted in compliance with GLP, Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics attained 73% degradation in 28 days. The 10-day window criterion is not applicable for complex substances where sequential degradation of the constituents takes place (OECD, 2006). The substance was therefore concluded to be readily biodegradable.

Executive summary:

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

 

Methods…….

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of approximately
24 ºC for 28 days. 

 

At the request of the Sponsor and following the recommendations of the International Standards Organisation (ISO, 1995), the test item was adsorbed onto a filter paper prior to subsequent dispersal in test media Using this method enables relatively small amounts of test item to be added accurately to the test vessels.

 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28 and by compound specific analysis on Days 0 and 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

 

Results…….

The test item attained 73% biodegradation after 28 days and therefore can be considered to be readily biodegradable.

Chemical analysis of the 100 mg/L test preparation at 0 hours showed a mean measured concentration of 102% of nominal was obtained. A decline in measured test concentration was observed on Day 28 to approximately 7% of nominal (93% loss over the test duration). 

 

The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to a combination of volatility of the test item and incorporation of the test item, or degradation products of the test item, into the microbial biomass. In such cases the micro-organisms present utilize carbon originating from the test item to increase their biomass by incorporation the carbon into new cells. This effectively removes the test item from the aqueous phase and hence reduces the apparent biodegradation of the test item as measured by oxygen consumption.

 

Chemical analysis of the abiotic test vessels on Day 0 showed a mean measured concentration of 108% of nominal was obtained. Analysis on Day 28 showed a mean measured concentration of 70% of nominal was obtained. Given the low total viable counts from the abiotic test vessels on Day 28 and the low biological oxygen consumption values from these vessels it was considered that this 35% loss of test item measured by chemical analysis on Day 28 occurred during sampling and analysis given the volatile nature of the test item.  

Description of key information

- The results from the ready biodegradability studies for several related substances in the relevant carbon number range for the registration substance ranged from about 10-70% after 28 days. Main factors influencing the results include the guideline/method followed, the dosing method and the viscosity/composition of the test substance. In general the biodegradability decreases as the viscosity (and hence carbon chain length/degree of branching) increases.

- Related substances containing relatively higher proportions of lower carbon number constituents meet the criteria for ready biodegradability.

- Overall 'Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear' is not considered to be readily biodegradable but is expected to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

In general terms, degradation can be seen to relate to the carbon range present in test material. This is consistent with water solubility limiting the rate of uptake by microorganisms.

In studies of any multi-constituent test substance, there will be uptake of the more bioavailable constituents first. If homologous series are present, it is possible that microorganisms will adapt to the general structural types present, but it is inevitable that rates will overall appear to be slower than for pure substances.

 

Therefore, where studies show high rates of degradation this can be considered to be indicative of the potential for high degradation in the environment, and such studies should be given higher weight in any overall assessment.

The 10-day window is not a necessary criterion for this type of substance, which is a UVCB and contains many structurally similar constituents (OECD, 2005).

 

A large number of tests for ready biodegradability have been conducted for a range of closely related GTL products (covering the carbon range from C18 to C57) with different viscosities, including the three grades 3, 4 and 8 cSt (at 40°C) that are relevant for supply in the EU.

The results from these ready biodegradability studies ranged from 11-65% after 28 days. The main factors influencing the results include the guideline/method followed, the dosing method and the viscosity/composition of the test substance.   

In general the biodegradability of the substances decreases as the viscosity (and hence carbon chain length/degree of branching) increases. In fact the lowest viscosity substance GTL Base Oil 3 (covering the carbon range from C18 to C30) meets the criteria for ready biodegradability, a view endorsed by the UK Centre for Environment, Fisheries and Aquaculture Science (Cefas) when they reviewed the data supplied for GTL Base Oil 3 under the OSPAR HOCNF (Oslo and Paris Commission Harmonised Offshore Chemical Notification Format) for their Offshore Chemical Notification Scheme (OCNS).

 

Moreover, degradation behaviour of structurally-similar substances in other screening studies is consistent with the conclusion that substances containing relatively higher proportions of lower carbon number constituents are readily biodegradable (ignoring the inapplicable 10-day window criterion):

 

-      Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics (covering the carbon range from C15 to C19) attained 73% biodegradation in 28 days in a ready biodegradation test conducted according to OECD 301F (Best, 2014a).

-      Hydrocarbons, , C18-C24, n-alkanes, isoalkanes, <2% aromatics (covering the carbon range from C18 to C24) attained 74% biodegradation in 28 days in a ready biodegradation test conducted according to OECD 301F (Best, 2014b). The same substance attained 52% biodegradation in a seawater biodegradation test conducted according to OECD 306 (Chemex, 2015).

 

-      GTL Gasoil (covering the carbon range from C8 to C26) attained 68% biodegradation in 28 days in a ready biodegradation test conducted according to OECD 301F.

 

Finally, BIOHCWIN (via the refined method described in the Chemical Safety Report) presents an easily applied method to estimate primary aerobic biodegradation half-lives in surface water. Complete removal (mineralisation) could take more time. Waste water treatment plants are likely to have higher concentrations of microorganisms and also high levels of other primary carbon sources. Therefore higher levels and rates of biodegradation in WWTP could be anticipated compared to half-lives predicted by BIOHCWIN. The measured results show that individually all constituents less than C20 are expected to be rapidly biodegradable. Constituents of ‘Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear’ above C20 may potentially be persistent or very persistent as defined in the REACH Technical Guidance, Chapter R.11, (ECHA, June 2017), but they will all degrade completely given sufficient time.

 

Conclusion:

Based on the available weight of evidence, ‘Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear’ is not considered to be readily biodegradable but is expected to be inherently biodegradable.