5-fluoro-2-methoxypyrimidin-4(3H)-one

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:

02 June 2005 to 01 July 2005

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:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The water treatment plant of Evreux (Evreux, France)
- Preparation of inoculum for exposure: The inoculum was prepared by initially decanting and sieving sewage sludge. The sludge was then centrifuged for 5 minutes, the supernatant was rejected and the pellet was re-dispersed in the mineral medium.
- Pretreatment: In order to wash out the dissolved organic carbon (DOC) and to lower the carbon organic content, the inoculum was preconditioned for 6 days before use. Air was bubbled through the inoculum during this preconditioning period. Sludge was maintained at 22 ± 2 °C and air was bubbled through the inoculum until use. Mineral medium (reconstituted water) was prepared using deionised water which contained no more than 5 % of the organic carbon content introduced by addition of the test or reference material and analytical grade reagents.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: A quantity of 10 mL of solution (a) was mixed with 800 mL of water, then 1 mL of solutions (b), (c) and (d) were added and the medium was made up to 1 litre with water.
Stock solution (a): KH2PO4 8.50 g, K2HPO4 21.75 g, Na2HPO4.2H2O 33.40 g and NH4Cl 0.50 g dissolved in water and made up to 1 litre. The pH of the solution was approximately 7.4.
Stock solution (b): CaCl2 27.50 g or CaCl2.2H2O 36.40 g dissolved in water and made up to 1 litre.
Stock solution (c): MgSO4.7H2O 22.50 g dissolved in water and made up to 1 litre.
Stock solution (d): FeCl3.6H2O 0.25 g dissolved in water and made up to 1 litre (in order to avoid having to prepare this solution immediately before use, one drop of concentrated HCl or 0.4 g EDTA disodium salt per litre was added).
- Test temperature: Between 20 and 26 °C in the test room
- pH: 7.32 measured in the mineral medium before the start of the test; 7.18 to 7.30 measured in the test flasks at the end of the test.
- pH adjusted: No
- Aeration of dilution water: Yes
- Suspended solids concentration: 20.0 mg/L of suspended solids (dry weight)
- Continuous darkness: Yes. The test was carried out in dark glass bottles fitted with dark glass stoppers and aeration tubes to reduce the quantity of light reaching the test suspensions.
- Other: In order to homogenise the solution, all test suspensions were agitated using magnetic stirrers.

TEST SYSTEM
- Culturing apparatus: Flasks. Parallel groups were prepared by adding 2.4 litres of mineral medium to each of the test flasks. Inoculum was added to provide a final concentration of 20.0 mg/L of suspended solids (dry weight) in three litres of suspension. The test flasks were aerated with CO2-scrubbed air overnight to purge the system of carbon dioxide. The test and reference materials were added, where appropriate, to the flasks to give a test and reference material concentrations of 10.0 mg of TOC per litre (71.9 mg of test material and 102.4 mg of sodium acetate). When all the substances have been added, the volume of the suspensions was made up to 3 litres.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Air was bubbled through each parallel at the rate of 30 - 100 mL/min (checked daily and reset if necessary) during the test. Carbon dioxide-scrubbed air was bubbled through the suspensions at the same rate for all preparations.
- Measuring equipment: CO2 was measured by titration of Ba(OH)2 traps
- Details of trap for CO2 and volatile organics if used: Test flasks were attached in parallel to a series of three wash bottles filled with 100 mL of 0.0125 M barium hydroxide solution (to trap any CO2 released from the test vessels).

SAMPLING
- Sampling frequency: Measurements of CO2 were made at the following times: days 1, 4, 6, 8, 11, 14, 18, 22, 25 and 29.
- Sampling method: For each measurement, the first wash bottle nearest to the test flask was disconnected and titrated with 0.05 M HCl, using phenolphthalein as an indicator. The remaining CO2 absorber bottles were connected to the test flasks so that the second wash bottle replaced the first one and an extra bottle containing fresh barium hydroxide solution was added to the far end of the series. For calculation purposes, it was assumed that the volume necessary to titrate untitrated wash bottle would be the same as the volume needed to titrate 100 mL of the Ba(OH)2 stock solution.
Each time CO2 was analysed, 100 mL of the barium hydroxide stock solution (used to fill the wash bottles) was titrated with the HCl solution in order to determine the maximum amount of acid required to titrate the wash bottles.
On the 28th day, 1 mL of concentrated hydrochloric acid was added to each test flask to stop the biodegradation and test flasks were aerated overnight to drive off the remaining carbon dioxide. A final CO2 analysis of all wash bottles was made on the 29th day, this final analysis being representative of biodegradation of the 28th day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Two flasks containing the inoculum
- Procedure control: One flask containing the reference material (at 10.0 mg/L of TOC) and inoculum
- Toxicity control: One flask containing the test material (at 10.0 mg/L of TOC), the reference material (at 10.0 mg/L of TOC) and inoculum

STATISTICAL METHODS
After the quantity of CO2 produced by biodegradation of the test material had been calculated, the percentage of biodegradation was determined using the formula:
% of biodegradation = (mg CO2 produced / ThCO2) x 100

ThCO2 = theoretical CO2 calculated to be produced from the known carbon content of the test material when fully degraded.
ThCO2 = mg TOC added to the test flask x 3.67, where TOC is the total organic carbon content of the test material and 3.67 is the conversion factor (44/12) from carbon to carbon dioxide.
Obtain the percentage degradation after a given time interval using the cumulative quantity of CO2 produced by the test material up to that time and the ThCO2 .
For calculation purposes, to determine percentages of biodegradation, it was assumed that the cumulative quantity of CO2 produced by a test or reference material replicate (or a toxicity control replicate) cannot be lower than the cumulative quantity of CO2 produced by the inoculum blank (average of the two replicates) at any time of the test.
In order to classify a test material as readily biodegradable, the biodegradation value of 60 % has to be reached in the 10-day window (the 10 days immediately following the attainment of 10 % biodegradation) within 28 days.

Reference substance:

acetic acid, sodium salt

Test performance:

All validity criteria were respected: Biodegradation values of test material replicates deviated by less than 20 % at the end of the test, biodegradation in the reference test was acceptable, the blank value (average of the two controls) was ≤70 mg of CO2/L at the end of the test (53.8 mg/L) and biodegradation in the toxicity control was 40 % (based on ThCO2) after 14 days (so at least 25 % within this period).

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

Biodegradation of the test material was 0 % (mean of the two flasks) over the test period.
The test material was suspected to be slightly toxic towards inoculum, based on cumulative quantity of CO2 released in the test solution.
At the end of the test, the cumulative quantity of CO2 produced in the test solution replicates was approximately 20 % lower than that of the control replicates. This indicates that the test material, at the concentration tested, had probably reduced the micro-organism efficiency to degrade the organic matter added to the test suspensions via the inoculum.
However, no significant inhibition of the inoculum activity by the test material was noted in the toxicity control, since biodegradation (based on cumulative quantity of CO2 produced by the test and reference materials) reached expected values without test material toxicity, i.e. 40 % of the TOC within 14 days and 48 % after 28 days.
Consequently, the suspected toxic effect of the test material towards inoculum was not considered as sufficiently marked to question the validity and the conclusion of the study.

Results with reference substance:

Biodegradation in the reference test was 77 % after 14 days (at least 6 0% within this period)

Table 1: Cumulative Percentage of Biodegradation

Day	Test Material			Reference Material	Toxicity Control
	Replicate 1	Replicate 2	Mean
1	0.00	0.00	0.00	14.54	7.02
4	0.00	0.00	0.00	36.87	18.28
6	0.00	0.00	0.00	59.60	31.25
8	0.00	0.00	0.00	72.48	36.54
11	0.00	0.00	0.00	76.23	38.66
14	0.00	0.00	0.00	77.33	39.51
18	0.00	0.00	0.00	76.93	40.91
22	0.00	0.00	0.00	77.58	42.29
25	0.00	0.00	0.00	77.43	43.41
28	0.00	0.00	0.00	75.78	47.68

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the conditions of this study, the biodegradation of the test material was 0 % at the end of the test period. Therefore, the test material was not readily biodegradable in the 28-day modified Sturm test.

Executive summary:

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301 B under GLP conditions.

The quantity of carbon dioxide (CO2) released by the degradation of the test material was determined using 4 different conditions. The conditions were: inoculum only (control); test material (10.0 mg/L TOC) and inoculum (test solution); reference material (sodium acetate, 10.0 mg/L TOC) and inoculum (procedure control); and test and reference materials (10.0 mg/L of TOC each) and inoculum (toxicity control).

The inoculum consisted of sewage sludge sampled from the aeration tank of a sewage treatment plant and aerated for 6 days. CO2 released by biodegradation was trapped in serial suspensions of Ba(OH)2 and measured by titration using a HCl solution. Measurements of CO2 were made regularly (at the beginning of the test and then every 1 to 5 days thereafter) over the 28-day test period.

Biodegradation of the test material was 0 % (mean of the two flasks) over the test period. The test material was suspected to be slightly toxic towards inoculum, based on cumulative quantity of CO2 released in the test solution. At the end of the test, the cumulative quantity of CO2 produced in the test solution replicates was approximately 20 % lower than that of the control replicates. This indicates that the test material, at the concentration tested, had probably reduced the micro-organism efficiency to degrade the organic matter added to the test suspensions via the inoculum. However, no significant inhibition of the inoculum activity by the test material was noted in the toxicity control, since biodegradation (based on cumulative quantity of CO2 produced by the test and reference materials) reached expected values without test material toxicity, i.e. 40 % of the TOC within 14 days and 48 % after 28 days. Consequently, the suspected toxic effect of the test material towards inoculum was not considered as sufficiently marked to question the validity and the conclusion of the study.

The validity criteria were respected: biodegradation of the test material replicates deviated by less than 20 % at the end of the test, the blank value (CO2 evolved uniquely from the breakdown of organic matter in inoculum) was ≤70 mg CO2/L of suspension at the end of the test, biodegradation of the reference material was 77 %, i.e. at least 60 %, after 14 days, and biodegradation in the toxicity control was 40 %, i.e. at least 25 %, after 14 days.

Under the conditions of this study, the biodegradation of the test material was 0 % at the end of the test period. Therefore, the test material was not readily biodegradable in the 28-day modified Sturm test.

Description of key information

Under the conditions of this study, the biodegradation of the test material was 0 % at the end of the test period. Therefore, the test material was not readily biodegradable in the 28-day modified Sturm test.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301 B under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The quantity of carbon dioxide (CO2) released by the degradation of the test material was determined using 4 different conditions. The conditions were: inoculum only (control); test material (10.0 mg/L TOC) and inoculum (test solution); reference material (sodium acetate, 10.0 mg/L TOC) and inoculum (procedure control); and test and reference materials (10.0 mg/L of TOC each) and inoculum (toxicity control).

The inoculum consisted of sewage sludge sampled from the aeration tank of a sewage treatment plant and aerated for 6 days. CO2 released by biodegradation was trapped in serial suspensions of Ba(OH)2 and measured by titration using a HCl solution. Measurements of CO2 were made regularly (at the beginning of the test and then every 1 to 5 days thereafter) over the 28-day test period.

Biodegradation of the test material was 0 % (mean of the two flasks) over the test period. The test material was suspected to be slightly toxic towards inoculum, based on cumulative quantity of CO2 released in the test solution. At the end of the test, the cumulative quantity of CO2 produced in the test solution replicates was approximately 20 % lower than that of the control replicates. This indicates that the test material, at the concentration tested, had probably reduced the micro-organism efficiency to degrade the organic matter added to the test suspensions via the inoculum. However, no significant inhibition of the inoculum activity by the test material was noted in the toxicity control, since biodegradation (based on cumulative quantity of CO2 produced by the test and reference materials) reached expected values without test material toxicity, i.e. 40 % of the TOC within 14 days and 48 % after 28 days. Consequently, the suspected toxic effect of the test material towards inoculum was not considered as sufficiently marked to question the validity and the conclusion of the study.

The validity criteria were respected: biodegradation of the test material replicates deviated by less than 20 % at the end of the test, the blank value (CO2 evolved uniquely from the breakdown of organic matter in inoculum) was ≤70 mg CO2/L of suspension at the end of the test, biodegradation of the reference material was 77 %, i.e. at least 60 %, after 14 days, and biodegradation in the toxicity control was 40 %, i.e. at least 25 %, after 14 days.

Under the conditions of this study, the biodegradation of the test material was 0 % at the end of the test period. Therefore, the test material was not readily biodegradable in the 28-day modified Sturm test.