Docosane

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 21 December 2005 and 18 January 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

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.

Key result

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 CO₂ analysis 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.

Description of key information

The results from the ready biodegradability studies for GTL Base Oil Distillates ranged from 11-67% 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

subtances decrease as the viscosity (and hence carbon chain length/degree of branching) increases. In fact, the lowest viscosity substances almost meet

the criteria for ready biodegradability. Overall GTL Base Oil Distillates is not considered to be readily biodegradable but is expected to be inherently

biodegradable.

This data is used for read across to Docosane.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

A large data set is available for whole product fractions of a range of GTL substances. The total solubility of constituents of these substances is <1 mg/l. Therefore, when the test substance is loaded into the medium at concentrations higher than this limit, the extent and rate of dissolution into water can be expected to influence the result of a ready biodegradation test. In such studies no measurements of the dissolved organic carbon from the substance are possible and therefore it cannot be known how much bioavailable (dissolved) constituents were present. The highest levels of reported degradation are those which should be given most weight in hazard assessment and risk characterisation.

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 GTL Base Oil Distillates substances with different viscosities, including the three grades (3, 4 and 8 cSt) relevant for supply in the EU.

 

The results from the ready biodegradability studies for GTL Base Oil Distillates ranged from 11-67% after 28d. 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 decrease as the viscosity (and hence carbon chain length/degree of branching) increases. In fact the lowest viscosity substances almost meet the criteria for ready biodegradability. Please refer to section 4.1.2.1.5 of the CSR for further details.

 

Overall GTL Base Oil Distillates is not considered to be readily biodegradable but is expected to be inherently biodegradable.

This data is used for read across to Docosane.