O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

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:

25 April 2017 to 24 May 2017

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:

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:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dispersed directly in mineral medium. An amount of test item (48.9 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 16.3 mg/L, equivalent to 10 mg carbon/L. A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK,
- 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 °C 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 deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Concentration of sludge: The suspended solids concentration was equal to 2.8 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

16.3 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral Medium: Solution a: KH2PO4 8.50 g/L, K2HPO4 21.75 g/L, Na2HPO4.2H2O 33.40 g/L and 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. In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per litre was added as a preservative. To 1 litre (final volume) of purified water (reverse osmosis purified and deionized water) was added the following volumes of solutions a to d: 10 mL of Solution a, 1 mL of Solution b, 1 mL of Solution c and 1 mL of Solution d.
- Test temperature: 22 to 24 °C
- pH: 7.4 to 7.7
- pH adjusted: Yes adjusted to pH 7.4 ± 0.2 on Day 0 using diluted hydrochloric acid or sodium hydroxide solution.
- Aeration of dilution water: 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.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels
- Number of culture flasks/concentration: 2
- Measuring equipment: 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.

ASSESSMENTS
- Observations: The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.
- pH Measurements: The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
- IC Analysis:
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. All samples were analysed for IC immediately. The remainder of all 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 analysed for IC using a Shimadzu TOC-LCSH TOC analyser. Samples (135 µL) were injected into the IC channel of the TOC analyser. 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 triplicate.
- IC/TC Ratio:
Samples (30 mL) were removed from the test material vessels on Day 0 prior to the addition of the test material in order to calculate the IC content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. IC/TC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
The samples were analysed for IC and TC using a Shimadzu TOC-VCPH TOC Analyser. Samples (50 µL) were injected into the TC and IC channels of the TOC analyser. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: An inoculated control, in duplicate, consisting of inoculated mineral medium.
- Procedure control: Containing the reference material (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L. The reference material, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference material directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 litres to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference material was inverted several times to ensure homogeneity of the solution.
- Toxicity control: The test material plus the reference material in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only). An amount of test material (48.9 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 16.3 mg test material/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

DATA EVALUATION
- Calculation of Carbon Content: The test material contains 61.27 % carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/L the total organic carbon present was 30 mg C. The theoretical amount of carbon present in the reference material, sodium benzoate (C6H5COONa) was calculated as follows:

[(No. of C atoms x Mol. Weight of C) / Mol. Weight of sodium benzoate] x 100
[(7 x 12.011) / 144.11] x 100 = 58.34 %

Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.

- Percentage Biodegradation: The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference materials before substitution into the following equation:

%ThCO2 (= % biodegradation) = [(mg IC in test flask – mg IC in control flask) / mg TOC added as test chemical] x 100

- Total CO2 Evolution: The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the following equation. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:

Total CO2 evolution (mg C/L) = mg IC in control x (100/ %C of CO2) x (1/test volume)

= mg IC in control x (100/27.29) x (1/3)

Reference substance:

benzoic acid, sodium salt

Test performance:

- The total CO2 evolution in the inoculum control vessels on Day 28 was 29.14 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 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.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

76

Sampling time:

28 d

Remarks on result:

other: St. dev. not reported.

Details on results:

- 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 decrease in all replicate vessels with the exception of procedure control replicate 1 and test replicate 2. This decrease was considered to be due to sampling/analytical variation. 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 76 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation 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 79 % biodegradation after 14 days and 83 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Results with reference substance:

Sodium benzoate attained 86 % biodegradation after 14 days and 97 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1: Percentage biodegradation values

Day	% Biodegradation
	Procedure control	Test material	Toxicity control
0	0	0	0
2	50	38	50
6	67	55	64
8	81	68	69
10	82	66	78
14	86	67	79
21	98	73	88
28	90	75	83
29*	97	76	83

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

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of this study the test item is considered to be readily biodegradable.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OCSPP 835.3110 under GLP conditions, in an aerobic aqueous medium.

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 and 24 °C for 28 days.

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

All validity criteria in the study were met. The toxicity control attained 79 % biodegradation after 14 days and 83 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Sodium benzoate (the positive control) attained 86 % biodegradation after 14 days and 97 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The test material attained 76 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. 

Under the conditions of this study the test material is considered to be readily biodegradable.

Description of key information

Under the conditions of this study the test material is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OCSPP 835.3110 under GLP conditions,in an aerobic aqueous medium. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 and 24 °C for 28 days.

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

All validity criteria in the study were met. The toxicity control attained 79 % biodegradation after 14 days and 83 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Sodium benzoate (the positive control) attained 86 % biodegradation after 14 days and 97 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The test material attained 76 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. 

Under the conditions of this study the test material is considered to be readily biodegradable.