N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in chloroform at 1000 mg/ 10 mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in chloroform prior to being dispensed and adsorbed to Whatman GF/A filter paper (70 mm diameter). The solvent on the filter paper was allowed to dry prior to test system application. High shear mixing was adopted to break up the filter paper and distribute it evenly through the test system.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: Stock solution prepared at 1000 mg/ 10 mL in chloroform, prior to dispensing and adsorption to filter paper.
- Final preparation of a solid: As described above.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a solid adsorbed to filter paper.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: N/A

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained for the initial experiment on 16 October 2017 and for the definitive test on 11 December 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Not reported
- Storage length: Not reported
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral 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 21 ºC 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 (Whatman) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC 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 2.8 g/L prior to use.
- Pretreatment: No
- Concentration of sludge: 2.8 g/L in prepared sludge "stock". Suspended solids added at a rate of 30 mg/L to the test vessels.
- Initial cell/biomass concentration: N/A
- Water filtered: Yes
- Type and size of filter used, if any: Whatman GF/A 70 mm

Duration of test (contact time):

29 d

Initial conc.:

5 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 3 L of mineral medium, prepared as per OECD 301B guidance innoculated with 30 mg/L activated sludge.
- Additional substrate: Not reported
- Solubilising agent (type and concentration if used): Chloroform used to prepare a stock solution of the test item - an aliquot of which was dispensed and adsorbed to filter paper. After the solvent had evaporated, the filter paper was added to the test system.
- Test temperature: 21 - 25 ºC
- pH: 7.4 ± 0.2
- pH adjusted: yes (dilute HCl or NaOH)
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Yes - CO2 free bubbled through test system at a rate of 30 - 100 mL/min.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes
- Other: N/A

TEST SYSTEM
- Culturing apparatus: 5 L test culture vessels
- Number of culture flasks/concentration: Inoculum control, Precedural control and Test Item flasks were prepared in duplicate. A single Toxicity control was prepared.
- Method used to create aerobic conditions: CO2 free air was bubbled through the test system
- Method used to create anaerobic conditions: N/A
- Measuring equipment: Shimadzu TOC-L/V analyser used to determine IC content in CO2 traps
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: 0.05 M NaOH
- Other: N/A

SAMPLING
- Sampling frequency: Day 0, 2, 6, 8, 10, 14, 21 28 and 29.
- Sampling method: 2 mL samples taken from CO2 traps for IC determination. On Day 28, 1 mL of HCl was added to each test vessel to drive off any inorganic carbonates and the test system resealed and aerated. Final 2 mL sample was taken on Day 29 from the CO2 traps.
- Sterility check if applicable: N/A
- Sample storage before analysis: Not reported
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated mineral medium and filter paper only
- Abiotic sterile control: No
- Toxicity control: Test item + reference item in inoculated mineral medium (15 mg C/L)
- Other: Reference item (sodium benzoate) in inoculated mineral medium (10 mg C/L)

STATISTICAL METHODS:
Statistical analysis of the Day 29 IC values for the inoculum control and test item vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Reference substance:

other: Sodium benzoate

Remarks:

Sigma-Aldrich, Batch # SLBT3039, Expiry: 09 May 2022

Preliminary study:

Preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO 10634, 1995) and in the published literature (Handley et al,2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper.

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

0 d

Parameter:

% degradation (inorg. C analysis)

Value:

2

Sampling time:

2 d

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

6 d

Parameter:

% degradation (inorg. C analysis)

Value:

1

Sampling time:

8 d

Parameter:

% degradation (inorg. C analysis)

Value:

1

Sampling time:

10 d

Parameter:

% degradation (inorg. C analysis)

Value:

5

Sampling time:

14 d

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

21 d

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Details on results:

The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Table 1       Percent biodegradation results

Day	% Biodegradation
	Procedural Control	Test Item	Toxicity Control
0	0	0	0
2	58	2	38
6	65	0	41
8	71	1	44
10	70	1	58
14	70	5	41
21	75	0	46
28	75	0	47
29*	66	0	52

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

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 0% biodegradation 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:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂ Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110.

 

An initial experiment was conducted at a concentration of 10 mg carbon/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25 % biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD guidelines, the test item would be classed as exhibiting inhibitory effects.

 

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item, at a reduced concentration of 5 mg C/L was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 to 25 °C for 28 days.

 

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation. 

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

 

The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

 

This biodegradation study is acceptable and satisfies the validity criteria outlined in OECD 301B.

Description of key information

Not readily biodegradable; OECD 301B, Best, N. (2018)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂ Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110.

 

An initial experiment was conducted at a concentration of 10 mg carbon/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25 % biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD guidelines, the test item would be classed as exhibiting inhibitory effects.

 

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item, at a reduced concentration of 5 mg C/L was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 to 25 °C for 28 days.

 

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation. 

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

 

The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

 

This biodegradation study is acceptable and satisfies the validity criteria outlined in OECD 301B.

[Type of water: freshwater]