N-(2-hydroxyethyl)dodecanamide

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:

13 June - 18 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

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)

Version / remarks:

January 1998

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, dark
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble to 100 mg/mL in methanol
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: insoluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto Whatman GF/A filter paper. High shear mixing was also applied to break up the filter paper containing the test item. 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.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Methanol stock prepared at 100 mg/mL. Test item applied filter paper was added to test vessels at a rate of 14.5 mg/L.
- Final preparation of a solid: See above.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test item applied to filter paper and added to test medium due to the poor water solubility of the test item.

OTHER SPECIFICS: n/a

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sludge collected 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: Continuous aeration
- 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* 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 3.0 g/L prior to use.
- Pretreatment: Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium (OECD 301) and 30 mL of inoculum and aerated overnight.
- Concentration of sludge: 30 mg suspended solids (ss)/L
- Initial cell/biomass concentration: n/a
- Water filtered: no
- Type and size of filter used, if any: n/a

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: Per 1 L of purified water: 10 mL KH2PO4 (8.50 g/L), K2HPO4 (21.75 g/L), Na2HPO4.2H2O (33.40 g/L), NH4Cl (0.50 g/L); 1 mL CaCl2 (27.50 g/L); 1 mL MgSO4.7H2O (22.50 g/L); 1 mL FeCl3.6H2O (0.25 g/L).
- Additional substrate: Innoculum, collected from the aeration stage of a sewage treatment plant 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* 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 3.0 g/L prior to use.
- Solubilising agent (type and concentration if used): Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent (methanol) prior to adsorption onto Whatman filter paper. High shear mixing was also applied to break up the filter paper containing the test item. 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.
- Test temperature: 21 - 24 ºC
- pH: 7.4 ± 0.2
- pH adjusted: yes - dilute HCl or NaOH solution, as required
- CEC (meq/100 g): not reported
- Aeration of dilution water: Yes
- Suspended solids concentration: 3.0 g/L
- Continuous darkness: yes
- Other: no

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels used to contain test solutions. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel 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 water.
- Number of culture flasks/concentration: Duplicate: treatment, reference and control test solutions. Singlicate: toxicity control (test item + reference item) test solution.
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel 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: n/a
- Measuring equipment: The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- 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 water.
- Other: no

SAMPLING
- Sampling frequency: Day 0, 2, 6, 8, 10, 14, 21, 28 and 29.
- Sampling method: 2 mL aliquots taken from CO2 trap (number 1) on Day 0, 2, 6, 8, 10, 14, 21, 28 and 29. 2 mL aliquots taken from second CO2 trap on Day 0 and 29. All samples were analyzed for IC immediately. The remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required. 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 analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with 3 replicates being used in the calculation/reported.
- Sterility check if applicable: n/a
- Sample storage before analysis: no
- Other: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, consisting of inoculated mineral medium plus a filter paper.
- Abiotic sterile control: no
- Toxicity control: yes, consisting of the test item on a filter paper plus the reference item (sodium benzoate) in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control
- Other: yes, procedure control containing the reference item (sodium benzoate).

STATISTICAL METHODS:

The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference items before substitution into the following equation:
%ThCO2 (% biodeg.) = ((mg IC in test flask - mg ICin control flask) / mg TOC added as test item) x 100
Total CO2 evolution (mg C/L) = mg IC in control x (100 / % C of CO2) x (1 / test volume) i.e. mg IC in control x (100 / 27.29) x (1 / 3)

Reference substance:

other: Sodium benzoate

Preliminary study:

From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, (1995)) and in the published literature (Handley et al, 2002) it was concluded that the best testable dispersion was found to be obtained when using the high shear mixing with solvent method of preparation.

However it was considered that the dimethylformamide was not fully evaporated from the filter paper in the initial experiment as the inorganic carbon values in the test item vessels were extremely high. Therefore further solubility work was conducted using methanol. A nominal amount of test item (1000 mg) was dissolved in methanol (10 mL) with the aid of shaking by hand for approximately 1 minute followed by ultrasonication for 3 minutes, and formed a clear colorless solution. An aliquot (450 μL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 30 minutes. The filter paper was then added to approximately 400 mL of mineral medium and subjected to high shear mixing (approximately 7500 rpm, 5 minutes). The volume was then adjusted to 3 liters with mineral medium. This formed a cloudy dispersion containing broken up pieces of filter paper and a layer of very small particles of test item on the surface. After 96 hours of magnetic stirring the appearance of the dispersion remained unchanged. Therefore this method of preparation was employed for the study.

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

69

Sampling time:

28 d

Details on results:

The test item attained 69 % biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. However, the test item has exhibited the potential for rapid biodegradation.

Results with reference substance:

Sodium benzoate attained 63 % biodegradation after 14 days and 66 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1       Percent Biodegradation

Day	% Biodegradation
	Procedural Control	Test Item	Toxicity Control
0	0	0	0
2	35	22	32
6	43	44	77
8	59	41	75
10	72	52	67
14	63	64	88
21	62	60	84
28	68	68	87
29*	66	69	83

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test item attained 69 % biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %.

Executive summary:

OECD 301B (2017) - The biodegradability of N-(2-hydroxyethyl) dodecanamide was assessed in an OECD 301B closed bottle test.

 

The test substance was exposed to a relatively low number of microorganisms present in mineral medium inoculated with activated sewage sludge, under aerobic conditions for a period of at least 28 days, with the test substance being the sole carbon and energy source.

Four test groups were prepared;

 

a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper (filter paper was used as a media for dispersing the insoluble teat item).

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 plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

 

Evolved CO₂was captured in 0.05 M sodium hydroxide traps from which 2 mL aliquots were removed periodically (Day 0, 2, 6, 8, 10, 14, 21, 28 and 29) throughout the test for inorganic carbon (IC) analysis.

 

The samples were analyzed for IC using either a Shimadzu TOC analyser with each analysis was carried out in at least triplicate.

 

Calculated results concluded that the test item attained 69 % biodegradation after 28 days.

 

Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %.

Description of key information

Readily biodegradable, but failing 10-day window; OECD 301B; Best, N. (2017)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Additional information

OECD 301B (2017) - The biodegradability of N-(2-hydroxyethyl) dodecanamide was assessed in an OECD 301B closed bottle test.

 

The test substance was exposed to a relatively low number of microorganisms present in mineral medium inoculated with activated sewage sludge, under aerobic conditions for a period of at least 28 days, with the test substance being the sole carbon and energy source.

Four test groups were prepared;

 

a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper (filter paper was used as a media for dispersing the insoluble teat item).

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 plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

 

Evolved CO₂ was captured in 0.05 M sodium hydroxide traps from which 2 mL aliquots were removed periodically (Day 0, 2, 6, 8, 10, 14, 21, 28 and 29) throughout the test for inorganic carbon (IC) analysis.

 

The samples were analyzed for IC using either a Shimadzu TOC analyser with each analysis was carried out in at least triplicate.

 

Calculated results concluded that the test item attained 69 % biodegradation after 28 days.

 

Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %.

[Type of water: freshwater]