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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test material was dissolved at 2000 mg/Lin dechlorinated water. The stock solution was agitated during 5 minutes. The pH of this stock solution was 6.45 after agitation.
Test solutions were prepared by further dilution of the stock solution with appropriate volumes of dechlorinated water and inoculum (i.e. activated sludge) to provide a geometric series of concentrations. The pH of the test solution of highest concentration (1000 mg/L) after addition of inoculum was 6.67.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The water treatment plant of Evreux (France).
- Method of cultivation: The inoculum was sampled one day before the test and maintained under agitation before use. As the sludge was not used immediately, 50 mL of sewage feed were added per litre just before agitation. Synthetic sewage feed was prepared following OECD recommendations. The culture was kept between 20 and 22 °C. The stock vessel was kept away from light by wrapping it in opaque material. Clean air was passed through the inoculum at the rate of 0.5 to 1 L/min until use. The pH: was checked before use and remained within acceptable limits (6.0 - 8.0) and therefore was not adjusted before the beginning of the test.
- Preparation of inoculum for exposure: The inoculum was left to settle and the supernatant rejected. It was then filtered (to remove the biggest particles) and washed once with dechlorinated water. Four samples of 50 mL were taken to determine the content in suspended matter (dry weight).
- Initial biomass concentration: The concentration of sludge (4.88 g/L) was adjusted to 4 g/L by addition of the appropriate volume of dechlorinated water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

280 ± 20 mg/Las CaCO3

Test temperature:

20 to 22 °C

pH:

6.0 - 8.0

Nominal and measured concentrations:

- Nominal concentrations: 10.0, 31.6, 100, 316 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Flasks. Solutions were prepared approximately every 15 minutes for test times of 3 hours. In order to verify the quality of the inoculum, the controls were prepared at the beginning (first control) and the end (second control) of the test.
- Material, size, headspace, fill volume: 500 mL. Each concentration was prepared separately by adding 16 mL of sewage feed to each of the flasks which were made up to 300 mL with dechlorinated water only in the case of the controls or a nominal mixture of dechlorinated water and test material (added by means of a stock solution) in the case of the test solutions. Each mixture was then made up to 500 mL by adding 200 mL of inoculum.
- Aeration: Flasks were aerated for three hours before oxygen measurements. The solutions were aerated at between 0.5 to 1 litre of air per minute using a Pasteur-pipette connected with a flexible tube to an air pump as an aeration device until the start of oxygen concentration measurements.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 4 g/L in the inoculum. In the test solutions, as 200 mL of inoculum were added in a final volume of 500 mL, the organic material concentration from inoculum was 1.6 g/L (± 10 %).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water was used with a hardness of 280 ± 20 mg/Las CaCO3.
- Particulate matter: The inoculum was filtered to remove the biggest particles

OTHER TEST CONDITIONS
- Adjustment of pH: No, the pH of inoculum remained within acceptable limits and therefore was not adjusted before the beginning of the test.
- Photoperiod: No special precautions were taken during the test
- Details on termination of incubation: After 3 hours of aeration/contact time, the content of the test flask was poured into the measuring apparatus and the oxygen concentration was determined for a period of approximately 10 minutes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Measurement of oxygen concentration of the solutions was determined using an oxygen probe placed into an opaque BOD (Biological Oxygen Demand) flask filled with the test or reference solution which was agitated with a magnetic stirrer. The oxygen probe was connected to a meter equipped with a chart recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Respiration rates of the test and reference solutions are presented in Table 1.
Inhibitions of the respiration rate were 0 % at 10.0, 31.6, 100 and 316 mg/L and 11 % at 1000 mg/L, relative to mean of the controls. Consequently, the highest concentration without significant effect (difference ≤ 15 %) on the respiration rate (NOEC), relative to mean of the controls, was ≥ 1000 mg/L.
As the difference in the respiration rate between the two controls was below 15 %, the test was therefore considered valid.

Results with reference substance (positive control):

The reference material was investigated at concentrations of 4, 12 and 36 mg/L. The EC50 was determined to be 10.8 mg/L with 95 % CI of 9.21 to 12.7 mg/L.
As the EC50 of 3,5-dichlorophenol was between 5 and 30 mg/L, the test was therefore considered valid.

Reported statistics and error estimates:

- Determination of the EC50 values
When for at least two concentrations, inhibition is > 0 % and < 100 %, the EC50 is calculated according to Probit analysis (i.e. Finney’s method, published by E. Weber, combined with Bliss's method). The 95 % confidence interval limits are calculated statistically according to Fieller's method.
When at only one concentration, inhibition is > 0 % and < 100 %, the EC50 is also calculated by Probit analysis. In this case, the highest concentration causing no inhibition and the lowest concentration producing 100 % inhibition are used as confidence limits.
If at all concentrations inhibition is 0 % or 100 %, the EC5O corresponds to the geometric mean of the highest concentration causing no inhibition and the lowest concentration producing 100 % inhibition. In this case, the highest concentration causing no inhibition and the lowest concentration producing 100 % inhibition can also be used as confidence limits.

Table 1: Respiration rates and percent inhibitions

Material	Concentration (mg/L)	Respiratory rate (mg O2/L/h)	Inhibition (%)
Control 1	0	36	-
Control 2	0	36	-
3,5-dichlorophenol	4	32	11
	12	13.5	63
	36	5.14	86
Test Material	10	36	0
	31.6	39	0
	100	36	0
	316	36	0
	1000	32	11

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 3-hour EC50 of the test material for the inhibition of respiration of activated sludge was >1000 mg/L.

Executive summary:

The effect of the test material on the respiration of activated sewage sludge was evaluated in a study conducted in accordance with the standardised guideline OECD 209 under GLP conditions.

Activated sludge (1.6 g/L) was exposed to the test material at nominal concentrations of 10.0, 31.6, 100, 316 and 1000 mg/L for 3 hours. Negative controls with activated sludge only were also run, as were positive controls in which activated sludge was exposed to 3,5-dichlorophenol at nominal concentrations of 4, 12 and 36 mg/L. 

The oxygen consumption (i.e. respiration rate) in test and reference solutions and in the control was measured for approximately 10 minutes, after aeration for 3 hours. The criterion measured was the EC50 of the test material.

Inhibitions of the respiration rate were 0 % at 10.0, 31.6, 100 and 316 mg/L and 11 % at 1000 mg/L, relative to mean of the controls. Consequently, the highest concentration without significant effect (difference ≤ 15 %) on the respiration rate (NOEC), relative to mean of the controls, was ≥ 1000 mg/L.

As the difference in the respiration rate between the two controls was below 15 % and the EC50 of 3,5-dichlorophenol was 10.8 mg/L (i.e. between 5 and 30 mg/L), the test was therefore considered valid.

Under the conditions of this study, the 3-hour EC50 of the test material for the inhibition of respiration of activated sludge was >1000 mg/L.

Description of key information

Under the conditions of this study, the 3-hour EC50 of the test material for the inhibition of respiration of activated sludge was >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The effect of the test material on the respiration of activated sewage sludge was evaluated in a study conducted in accordance with the standardised guideline OECD 209 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Activated sludge (1.6 g/L) was exposed to the test material at nominal concentrations of 10.0, 31.6, 100, 316 and 1000 mg/L for 3 hours. Negative controls with activated sludge only were also run, as were positive controls in which activated sludge was exposed to 3,5-dichlorophenol at nominal concentrations of 4, 12 and 36 mg/L. 

The oxygen consumption (i.e. respiration rate) in test and reference solutions and in the control was measured for approximately 10 minutes, after aeration for 3 hours. The criterion measured was the EC50 of the test material.

Inhibitions of the respiration rate were 0 % at 10.0, 31.6, 100 and 316 mg/L and 11 % at 1000 mg/L, relative to mean of the controls. Consequently, the highest concentration without significant effect (difference ≤ 15 %) on the respiration rate (NOEC), relative to mean of the controls, was ≥1000 mg/L.

As the difference in the respiration rate between the two controls was below 15 % and the EC50 of 3,5-dichlorophenol was 10.8 mg/L (i.e. between 5 and 30 mg/L), the test was therefore considered valid.

Under the conditions of this study, the 3-hour EC50 of the test material for the inhibition of respiration of activated sludge was >1000 mg/L.