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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2005 to 24 June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: As the test material was not found to be toxic at 100 mg/L nominal, chemical analysis was undertaken to measure the concentration of the test material in this solution.
- Sampling method: At 0 hours, a sample was taken from the limit test solution (100 mg/L nominal) before addition of algae and stored until analysis. At 24, 48 and 72 hours, samples were taken from each limit test solution replicate, pooled (5 mL in total) and kept until analysis. Before analysis, the samples containing algae were centrifuged (after thawing and mixing) and the supernatant was analysed separately to verify the test material level in solution throughout the test. The samples with algae were centrifuged at 4000 rpm, 15 min and +4 °C. Then each sample was diluted (20-fold) in mobile phase to achieve concentrations in the range 0.1 to 10 mg/L.
Further samples were also taken at 24, 48 and 72 hours from the limit test solution which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test material concentration throughout the test.
- Sample storage conditions before analysis: The test material sample solution was kept at -20 °C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solutions for the limit and range-finding tests were prepared by dissolving the test material directly in LC reconstituted water.
- Quantity of test material: 100 mg
- Volume of test water: 1000 mL
- Concentration of test material: 100 mg/L
- Duration of agitation: 5 minutes
After agitation, the stock solution was immediately used to prepare the test solutions. Test solutions were prepared by further dilution of the stock solution with LC reconstituted water to provide a geometric series of concentrations: 0, 0.01, 0.1, 1, 10 and 100 mg/L.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain No.: CCAP 276/20
- Source (laboratory, culture collection): Cultured at the testing facility, originally obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN, Argyll, PA37 1QA, Scotland.
- Age of inoculum (at test initiation): Algae are cultivated for 7 days before harvesting for use in a new culture.
- Method of cultivation: The algae are cultured under sterile conditions and maintained at exponential growth rate. Each weekly culture is prepared under sterile conditions using autoclaved medium while week-old cell cultures are checked for contamination before being transferred to fresh medium.

ACCLIMATION
- Acclimation period: Algae are cultivated for 7 days before harvesting for use in a new culture
- Culturing media and conditions (same as test or not): Test media was the same as culture media
- Any deformed or abnormal cells observed: No data

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

The hardness of the culture medium is approximately 34 ± 17 mg/L as CaCO3

Test temperature:

24.2 to 25.0 °C

pH:

6.29 to 8.13

Conductivity:

<10 μS/cm

Nominal and measured concentrations:

- Limit test: 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: Stoppered with cotton wool
- Material, size, headspace, fill volume: 100 mL
- Aeration: Test solutions were not aerated during the test but the cultures were constantly agitated
- Initial cell density: 1 x 10^4 cells/mL
- Control end cell density: Mean ln number of cells = 13.38
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per analysis solution (limit concentration without algae): 1 replicate

GROWTH MEDIUM
- Standard medium used: Yes, LC reconstituted water
- Detailed composition: A primary stock solution is made up every month from which a final culture medium is prepared. This solution is autoclaved at which point it has a stock life of one week. Before use, the pH is verified at 7.5 ± 0.3 or adjusted until this pH is obtained. The hardness of this solution is approximately 34 ± 17 mg/L as CaCO3.
The compositions of the component solutions and their final concentrations are as follows (all solutions are made up with injectable grade deionised water):
Solution No. 1: Ca(NO3)2.4H2O 40 mg/L
Solution No. 2: KNO3 100 mg/L
Solution No. 3: MgSO4.7H2O 30 mg/L
Solution No. 4: K2HPO4 40 mg/L
Solution No. 5 (trace element solutions): CuSO4.5H2O) 15 µg/L, (NH4)6Mo7O24.4H2O 30 µg/L, ZnSO4.7H2O) 30 µg/L, CoCl2.6H2O 30 µg/L, Mn(NO3)2.4H2O 30 µg/L, C6H8O2.H2O 30 µg/L and H3BO3 30 µg/L
Solution No. 6: C6H5FeO7 0.616 mg/L, FeSO4.7H2O 0.3125 mg/L and FeCl3.6H2O 0.3125 mg/L

TEST MEDIUM / WATER PARAMETERS
- Conductivity: <10 µs/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was controlled daily in a flask containing test water but no algae, run alongside the test. The pH values of the control and the limit test concentration (100 mg/L) were measured at the beginning and the end of the test. Water hardness was measured once in LC reconstituted water before the start of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No. The pH of all test solutions remained within the range 6.0 ± 0.2 to 9.0 ± 0.2 after preparation and there was therefore no adjustment of pH before addition of the algae.
- Photoperiod: Continuous illumination
- Light intensity and quality: A set of 15 and 30 W Osram Fluora® fluorescent tubes between "white" and "daylight" type (spectral range 400 to 700 nm) were set approximately 40 cm above the cultures. The colour temperature of the fluorescent lamps is between 4400 and 5000 K. The lighting varied between 6910 and 7680 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: For the observation times (24, 48 and 72 hours), cell numbers were determined using a Malassez cell counter.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.01, 0.1, 1 and 10 mg/L (two replicates per concentration)
- Results used to determine the conditions for the definitive study: Yes. Inhibitions of the growth rate and growth at 0.01, 0.1, 1 and 10 mg/L were 0 % relative to the control after 72 hours.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

LIMIT TEST
Inhibitions of the growth rate and growth at 100 mg/L nominal were 0 %, relative to the control, after 72 hours.
The No Observed Effect Concentration (NOEC) after 72 hours was ≥ 100 mg/L nominal (p = 0.05) based on both the specific growth rate data and biomass data.
As the test material was not found to be toxic at 100 mg/L nominal, chemical analysis was undertaken to measure the concentration of the test material in this solution.
Measured concentrations in the solutions with and without algae were within ± 20 % of the nominal value (100 mg/L) throughout the test. The above-mentioned study results could therefore be based on nominal concentrations.
As no inhibition of the algal growth was observed at 100 mg/L and the measured concentrations of the test material were within ± 20 % of this nominal value throughout the test, the study was considered complete.

VALIDITY CRITERIA
All validity criteria of the study were respected. The cell concentration in the control cultures increased by a factor of at least 16 within 3 days. The coefficient of variation of average growth in replicate control cultures was ≤ 15 %.
Additionally, the coefficient of variation of daily growth rates in replicate control cultures during the course of the test (days 0-1, 1-2 and 2-3) did not exceed 35 %.

Reported statistics and error estimates:

- Determination of the ErC50 and the EbC50
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 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 EC50 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.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72-hour ErC50 was > 100 mg/L, for Scenedesmus subspicatus. The NOEC at 72 hours was ≥ 100 mg/L.

Description of key information

Under the conditions of this study, the 72-hour ErC50 was > 100 mg/L, for Scenedesmus subspicatus. The NOEC at 72 hours was ≥ 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A study was conducted to assess the effect of the test material on the growth of Scenedesmus subspicatus under static conditions in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a range-finding test, a limit test was conducted at 100 mg/L to assess the toxicity of the test material in algae. Three replicate solutions of algae with an initial dilution of 1 x 10^4 cells/mL were exposed to a nominal concentration of 100 mg/L and a further group of six replicate algal solutions without test material was used as the control. The growth of the cell culture in each flask was monitored by counting the number of cells at 24, 48 and 72 hours.

Inhibitions of the growth rate and growth at 100 mg/L nominal were 0 %, relative to the control, after 72 hours. All validity criteria of the study were fulfilled therefore the test was considered valid. As the test material was not found to be toxic at 100 mg/L nominal, chemical analysis was undertaken to measure the concentration of the test material in test solutions and measured concentrations were within ± 20 % of the nominal value (100 mg/L) throughout the test.

Under the conditions of this study, the 72-hour ErC50 was > 100 mg/L, for Scenedesmus subspicatus. The NOEC at 72 hours was ≥ 100 mg/L.