Flucytosine

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-05-27 - 2004-08-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Flucytosine

EC Number:

217-968-7

EC Name:

Flucytosine

Cas Number:

2022-85-7

Molecular formula:

C4H4FN3O

IUPAC Name:

flucytosine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Please refer to Test material information

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: Preliminary test samples were prepared, analysed initially and then after storage in sealed glass vessels at ambient temperature in light and dark conditions for approximately 24 hours (equivalent to the period of media renewal). The test material was stable in the test medium for the period of media renewal.
- Solubility and stability of the test substance in the solvent/vehicle: The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 hours (fresh media), 24 and 96 hours (old media). Analysis of the test preparations at 0, 24 and 96 hours (see Appendix 1) showed measured test concentrations to be near nominal.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: see below
- Final preparation of a solid: the test material was dissolved directly in dechlorinated tap water. An amount of test material (4000 mg) was dissolved in dechlorinated tap water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 4000 mg/l stock solution. An aliquot (500 ml) of this 4000 mg/l stock solution was then added to a final
volume of 20 litres of dechlorinated tap water to give the 100 mg/l test concentration. This method of preparation was conducted in duplicate to give replicates R1 and R2 . The stock solution was inverted several times to ensure adequate mixing and homogeneity.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
see above

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Range finding test: 1.0, 10 and 100 mg/L; Definitive test: 100 mg/mL
-- Physico-chemical measurements: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH was measured using a WTW pH 320 pH meter, the dissolved oxygen concentration was measured using a YSI 550 dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
-- Verification of test concentrations: Water samples were taken from the control and each replicate test vessel at 0 hours (fresh media), 24 and 96 hours (old media) for quantitative analysis. Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20°C for further analysis if necessary.
- Sample storage conditions before analysis: -20°C

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test the test material was dissolved directly in dechlorinated tap water. An amount of test material (4000 mg) was dissolved in dechlorinated tap water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 4000 mg/l stock solution. An aliquot (500 ml) of this 4000 mg/L stock solution was then added to a final
volume of 20 litres of dechlorinated tap water to give the 100 mg/L test concentration. This method of preparation was conducted in duplicate to give replicates R1 and R2 . The stock solution was inverted several times to ensure adequate mixing and homogeneity.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): A test sample was tested for stability without prior mixing (sonication) the test sample bottle to assess for losses due to adsorption and/or insolubility.The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout (Oncorhynchus mykiss)
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): mean standard length of 4.5 cm (sd = 0.3) at the end of the definitive test.
- Weight at study initiation (mean and range, SD): mean weight of 1.31 g (sd = 0.37) at the end of the definitive test.
- Method of breeding:
- Maintenance of the brood fish: in-house since 14 July 2004. Fish were maintained in a glass fibre tank with a "single pass" water renewal system.

ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 11 August 2004 to 23 August 2004.
- Acclimation conditions (same as test or not): The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): There was zero mortality in the 7 days prior to the start of the test

FEEDING DURING TEST
none

Study design

Test type:

semi-static

Water media type:

freshwater

Remarks:

Laboratory tap water, dechlorinated and softened

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

approximately 100 mg/L as CaCO3

Test temperature:

approximately 14°C

pH:

mean pH = 7.6 prior to dechlorination and softening. There was no treatment related difference for pH.

Dissolved oxygen:

there was no treatment related difference for oxygen concentration

Conductivity:

mean 439 µS/cm prior to dechlorination and softening

Nominal and measured concentrations:

Analysis of the test preparations at 0, 24 and 96 hours (see Appendix 1) showed measured test concentrations to be near nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: see below
- Type (delete if not applicable): The test vessels were covered to reduce evaporation
- Material, size, headspace, fill volume: 20 litre glass exposure vessels
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate):
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 100 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 1.700 mg C/L prior to dechlorination and softening
- Pesticides: <0.010 µg/L prior to dechlorination and softening
- Chlorine: 27.000 mg Cl/L prior to dechlorination and softening
- Alkalinity: 113.000 mg HCO3/L prior to dechlorination and softening
- Ca/Mg ratio: 51.000 mg Ca/L; 8.000 mg Mg/L prior to dechlorination and softening
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Range finding study test concentrations: 1.0, 10 and 100 mg/L
- Definitive test test concentration: 100 mg/l
- Results used to determine the conditions for the definitive study: After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. There were no sub-lethal effects of exposure during the range-finding test. The results showed no mortalities at the test concentrations of 1.0, 10 and 100 mg/L.
Based on this information, a single test concentration, in duplicate, of 100 mg/L was selected for the definitive test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: based upon mortalities and any sub- lethal effects

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: based upon mortalities and any sub- lethal effects

Remarks on result:

other:

Remarks:

nominal test conc. analytically verified

Details on results:

There were no sub-lethal effects of exposure observed in the test.

Results with reference substance (positive control):

-

Reported statistics and error estimates:

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the LC 50 values in terms of the nominal test concentrations only.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Range-finding Test

There were no sub-lethal effects of exposure during the range-finding test.

The results showed no mortalities at the test concentrations of 1.0, 10 and 100 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC 50 of greater than 100 mg/L. Correspondingly the No Observed Effect Concentration was 100 mg/L. No sub-lethal effects were observed.
Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.