N-(2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

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:

Experimental start date: 23 October 2012 Experimental end date 22 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N4-Benzoylcytosine

EC Number:

628-907-2

Cas Number:

26661-13-2

Molecular formula:

C11H9N3O2

IUPAC Name:

N4-Benzoylcytosine

Specific details on test material used for the study:

Identification: N-Benzoyl Cytosine
Description: Off white powder
Batch: NBC-5-11001
Purity: 100%
Expiry / Retest Date: Not supplied
Storage Conditions: Room temperature, over silica gel, in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Test Species
A mixed population of activated sewage sludge micro-organism was obtained on 22 October 2012 from aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, Uk, which treats predominatly domestic sewage.

Preparation Of Inoculum
The activated sewage sludge sample was washed three times by settlment and resuspension 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 continous 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 sludge by suction through pre-weighed GF/A filter paper (rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successuve time with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an ivern 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. The suspended solids concentration was equal to 2.2 g/L prior to use

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

16.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Mineral Medium
The mineral medium used in this study was that reccmended in the OECD guidelines.

Experimental Preparation
For the purpose of the test, the test item was dispersed directly in mineral medium
An amoun tof test item *48.9 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (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 carbon/L was employed in the test following the recommendations of the test guidelines.
Data from the inoculum control vessels was shared with similar concurrent studies.

Reference Item
For the purpose of the test, a reference item sodium benzoate (C6H5COONa), was used to prepare the procedure control vessles. An initial stock solution of 1000 mg/L was prepared by dissolving the refernce item directly in mineral medium with the aid of ultrasonication for approximately 2 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the colume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted sevral times to ensure homogeneity of the solution.
Data from the inoculum control vessels was shared with similar concurrent studies.

Toxicity Control
For the purpose of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic affect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (48.9 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (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 liters to give a final concentration of 16.3 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of test system
The following test preparation were prepared and inoculated in 5 liters test culture vessels each containing 3 liters of solution;
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test ietm 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).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at 21 to 22 °C in darkness.
Approximately 24 hours prior to additon of the test item and reference items th vessels were filled with 2400 mL of mineral medium and 40.9 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.
The test vessels were sealed and CO2 free air bubbled through the solution at a rate of 30 to 100 mL/min per vessle and stirre continuously by magnetic stirrer. The CO2 free air was produced by passing compressed air though a glass column containing self indicating soda line (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.

Validation Criteria
The results of the degradation test are considered valid if in the same test the reference item yields ≥60% degradation by day 14.
The test item may be considered to be readily biodegrable if ≥ 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain ≥ 25% degradation by day 14 for the test item to be considered as non iinhibitory.
The test is considered valid if the difference of the extreme of replicates values of production of CO2 aat the time the plateau is reached, at the end of the test or at the at the end of the 10 day window, as appropiatem is less than 20 %.
The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.
The IC content of the test item suspension in the mineral medium at the beginning of the test item should be <5 % of the TC.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

94

Sampling time:

28 d

Details on results:

The total CO2 evolution in the inoculum control vessels on day 28 was 31.33 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item 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 results of the inorganic carbon analysis of samples form the first absorber vessels on day 29 showed a decrease in all replicate vessels with the exception on inoculum control replicates R2 and procedure control replicate R2. This decrease was considered to be dure to sampling/ analytical varation.
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 occured.
The toxicity control attained 91% degradation after 14 days and 85 % degradation after 28 days thereby confirming that the test item 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 sampling/ analytical variation.

BOD5 / COD results

Results with reference substance:

Sodium Benzoate attained 101% degradation after 14 days and 109 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be dure to sampling/ analytical variations.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 94% degredation after 28 days and satisfied the 10 day window validation criterin, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of the OECD Guideline No 301B.

Executive summary:

Introduction

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 Chemical (1992) No. 301B, "Ready Biodegrability: CO₂ Evolution Test" referenced as Method C.4 -C of Commission Regulations (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Method

The test item, at 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 21 to 22 °C for 28 days.

The degradation 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 purpose.

Results

The test item attained 94% 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 item can therefore be considered to be readily biodegradable under the strict terms and conditions of the OECD Guideline No 301B.