Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

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:

2008/06/03-2008/07/03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge:
A mixed population of activated sewage sludge micro-organisms was obtained on 2 June 2008 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture:
Not applicable.

- Method of cultivation:
Not applicable.

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

- Storage length:
Not applicable, inoculum 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 approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.1 g/l prior to use.

- Pretreatment:
Not stated.

- Concentration of sludge:
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l.

- Initial cell/biomass concentration:
Not stated.

- Water filtered:
yes, Reverse osmosis purified and deionised water

- Type and size of filter used, if any:
(Elga Optima 15+ or Elga Purelab Option R-15 BP)

Duration of test (contact time):

28 d

Initial conc.:

16.1 mg/L

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
* Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

- Additional substrate:
None.

- Solubilising agent (type and concentration if used):
None.

- Test temperature:
The test was carried out in a temperature controlled room at approximately 21 deg C.

- pH:
The pH of the test preparations on day 28 was determined to be between 7.6 and 7.8.

- pH adjusted:
No

- CEC (meq/100 g):
Not stated.

- Aeration of dilution water:
Not stated.

- Suspended solids concentration:
2.1 g/l prior to use. Each test vessel was inoculated with prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.

- Continuous darkness:
Yes

- Other:


TEST SYSTEM
- Culturing apparatus:
5 litre glass culture jars

- Number of culture flasks/concentration:
Control: 2 replicates
Sodium Benzoate: 2 replicates
Test material: 2 replicates
Test material + sodium benzoate: One vessel only

- Method used to create aerobic conditions:
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.

- Method used to create anaerobic conditions:
Not applicable.

- Measuring equipment:
CO2 analysis:
Samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser.
Dissolved Organic Carbon analysis:
Samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser.

- Test performed in closed vessels due to significant volatility of test substance:
Not applicable.

- Test performed in open system:
The culture vessels were sealed.

- Details of trap for CO2 and volatile organics if used:
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.

- Other:

SAMPLING
- Sampling frequency:
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, 12, (replicate control and standard material samples only) 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately.
Dissolved Organic Carbon 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.
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.
DOC analysis of the test material dispersions after dosing was not possible due to the limited solubility of the test material in culture medium.

- Sampling method:
Not stated.

- Sterility check if applicable:
Not stated.

- Sample storage before analysis:
The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 12, 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank:
A control, in duplicate, consisting of inoculated culture medium.

- Abiotic sterile control:
Not stated.

- 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 (161 ml) of the test material stock dispersion 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 16.1 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.


STATISTICAL METHODS:
Determination of carbon content
The test material contains 62.18% carbon and so for a concentration of 10 mg C/l (a total of 48.3 mg of test material in 3 litres) the total organic carbon present was 30 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%

=((7x12.011)/144.11) x 100 = 58.34%

Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.

Percentage degradation
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.

%ThCO2(=%degradation) = ((mg IC in test flask - mg IC in control)/mg TOC as test material) x 100%

The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Percentage degradation = [1-(mg DOC in test flask on Day 28 / mg DOC in test flask on Day 0)] x100%

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 = mg IC in control x (100/5C of CO2) x (1/test volume)
= mg IC in control x (100/27.29) x (1/3)

Reference substance:

benzoic acid, sodium salt

Remarks:

Sodium benzoate (C6H5COONa)

Preliminary study:

No preliminary study performed.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 31.07 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 (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

Parameter:

% degradation (CO2 evolution)

Value:

76

Sampling time:

10 d

Remarks on result:

other: Satisfied the 10 day window validation criteria

Parameter:

% degradation (CO2 evolution)

Value:

72

Sampling time:

28 d

Details on results:

See overall remarks section.

Results with reference substance:

Sodium benzoate attained 77% degradation after 14 days and 75% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in degradation between Days 14 and 28 was considered to be due to normal biological, sampling and/or analytical variation.

Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.

 

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.

 

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. 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 test material attained 70% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

 

The toxicity control attained 76% degradation after 14 days and 67% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The slight decrease in degradation between Days 14 and 28 was considered to be due to normal biological, sampling and/or analytical variation.

 

Sodium benzoate attained 77% degradation after 14 days and 75% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in degradation between Days 14 and 28 was considered to be due to normal biological, sampling and/or analytical variation.

 

Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 102% and 92% respectively for Replicates R1 and R2. 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 sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.

 

Observations made throughout the test period (see Table 6) 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.

 

Days 0 showed the contents of the test material vessels to be cloudy light brown dispersions with no undissolved test material visible and on Day 6 the contents of the test material vessels were cloudy light brown dispersions with few particles of test material visible dispersed throughout.

On Days 13, 20 and 27 the contents of the test material vessels were observed to be cloudy light brown dispersions with no undissolved test material visible

 

The contents of the toxicity control vessel on Day 0 were a cloudy light brown dispersion with no undissolved test material or standard material visible whilst on Day 6 they were a cloudy light brown dispersion with a few particles of test material visible throughout and no undissolved standard material visible. On Days 13, 20 and 27 the contents were observed to contain slightly cloudy light brown dispersions with no undissolved test or standard material visible.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 70% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. 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; CO₂Evolution 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 10mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

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 70% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

Readily biodegradable, EU Method C.4-C, OECD 301B, EPA OPPTS 835.3110, Clarke (2008)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In the key study (Clarke 2008), the ready biodegradability of the test material was assessed 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).

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality as described in Klimisch et al. (1997).

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

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.

The test material attained 70 % degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % degradation must be attained within 10 days of the degradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.