Fatty acids C18-(unsaturated) lithium salts

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The preliminary investigational work was conducted on 12 January 2006 and the definitive test was conducted between 14 February 2006 and 15 March 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

LiTOFA

IUPAC Name:

LiTOFA

Details on test material:

Sponsor's identification :Reaction Salt of lithium hydroxide and tall oil fatty acids (LiTOFA)
Description : off white solid
Batch number: LiTOFA_aO1
Date received: 20 October 2005
Storage conditions :room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK

- Laboratory culture: Not applicable

- Method of cultivation: Not applicable

- Storage conditions: Not applicable, as used on day of collection

- Storage length: Not applicable, as used on day of collection

- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.

- Pretreatment: Not applicable

- Concentration of sludge: The suspended solids was equal to 4.0 g/l prior to use.

- Initial cell/biomass concentration: Not reported

- Water filtered: Prior to filtration through paper, rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

- Type and size of filter used, if any: pre-weighed GF/A filter paper using a Buchner funnel

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

other: mg of carbon per litre

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:

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

pH = 7.4

Solution b CaCl2 27.50 g/l
Solution c MgSO4.7H2O 22.50 g/l
Solution d FeCl3.6H2O 0.25 g/l

To 1 litre (final volume) of purified water was added the following volumes of solutions a – d.

10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

- Additional substrate: not applicable

- Solubilising agent (type and concentration if used): not used. For the purpose of the test the test material was dissolved directly in culture medium

- Test temperature: 20°C

- pH: 7.8 - 7.9

- pH adjusted: The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter

- CEC (meq/100 g): not stated in report

- Aeration of dilution water:

- Suspended solids concentration: The suspended solids was equal to 4.0 g/l prior to use.

- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 5 litre glass culture vessels each containing 3 litres of solution
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).

Number of culture flasks/concentration: Two (in duplicate)

- Method used to create anaerobic conditions: not applicable

PREPARATION OF TEST SYSTEM
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 22.5 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, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12 and 18 were stored at approximately 20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that degradation of the test material had met the 10-Day window validation criterion given in the OECD Guidelines 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, Shimadzu TOC-VCSH TOC analyser and an Ionics 1555B TOC analyser. Samples (300, 50 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 to CO2 by orthophosphoric acid using zero grade air or nitrogen (oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
On Days 0 and 28 samples (20 ml) were removed from all culture 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. 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.


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 aliquot (133 ml) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 13.3 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.


STATISTICAL METHODS: Not applicable

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Preliminary study:

Not conducted

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

83

Sampling time:

28 d

Remarks on result:

other: Satisfied 10-day window

Details on results:

The test material attained 83% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 82% degradation after 14 days and 77% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in standard material replicate R1 first absorber vessel coupled with the decrease in inorganic carbon within control replicates R1, R2 and standard material replicate R2 on Day 29 resulted in an increase in the percentage degradation value for the standard material from 77% on Day 28 to 79% on Day 29.

Any other information on results incl. tables

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in standard material replicate R₁and test material replicate R₂, a decrease in control replicates R₁and R₂, standard material replicate R₂, test material replicate R₁and the toxicity control vessel. 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 coupled with the decrease in inorganic carbon within control replicates R₁and R₂ and test material replicate R₁resulted in a decrease in the percentage degradation value for the test material from 83% on Day 28 to 82% on Day 29.

The toxicity control attained 87% degradation after 14 days and 90% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The decrease in inorganic carbon in the first absorber vessel on Day 29 coupled with the decrease in inorganic carbon within control replicates R₁and R₂resulted in a decrease in the percentage degradation value for the toxicity control from 90% on Day 28 to 86% on Day 29.

Sodium benzoate attained 82% degradation after 14 days and 77% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in standard material replicate R₁first absorber vessel coupled with the decrease in inorganic carbon within control replicates R₁, R₂ and standard material replicate R₂on Day 29 resulted in an increase in the percentage degradation value for the standard material from 77% on Day 28 to 79% 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 from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), gave percentage degradation values of 97% and 100% respectively for the test material Replicates R₁and R₂and 99% for the toxicity control. Sodium benzoate attained 101% and 98% degradation respectively for Replicates R₁and R₂calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of test material/sodium benzoate into the microbial biomass prior to degradation, and hence CO₂evolution 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 light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown slightly cloudy dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown slightly cloudy dispersion with no undissolved standard material or test material visible.

Percentage degradation values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	18	0	13
2	44	0	23
3	55	19	31
6	71	53	67
8	75	65	74
10	76	70	81
14	82	83	87
16	81	79	87
20	87	80	88
22	82	83	89
24	80	84	92
27	80	85	92
28	77	83	90
29*	79	82	86

*Day 29 values corrected to include any carry-over of CO₂ detected in Absorber 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 83% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test material 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).

Methods.

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

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.

The test material attained 83% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.