Flucytosine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-05-25 - 2004-08-25

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

Two temperatures in test vessels were measured to be 0.2°C above the 21 ± 1°C range at 0 hours. This was considered not to affect the results as no adverse effects of exposure were observed in the control daphnids throughout the test.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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
- Stability under test conditions: Throughout the duration of the test the test preparations were observed to be clear, colourless solutions.
- 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 and 48h. In addition 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 test samples have been shown to be stable in the test medium. The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of the definitive test the test material was dissolved directly in reconstituted water.
- Final preparation of a solid: An amount of test material (200 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 2 minutes and the volume adjusted to 2 litres to give the 100 mg/L test concentration. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Sampling and analysis

Analytical monitoring:

yes

Remarks:

at 0 and 48h

Details on sampling:

- Concentrations:
- Sampling method: Water samples were taken from the control (replicates R1 – R2 pooled) and 100 mg/L test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L. The test material was dissolved directly in water.
An amount of test material (100 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 2 minutes and the volume adjusted to 1 litre to give a 100 mg/L test concentration. Serial dilutions were then performed to prepare the 1.0 and 10 mg/L test concentrations. The prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. Due to suspected light sensitivity, the test material was weighed out and diluted under a non-actinic light source for the range-finding test. However, stability analysis showed the test material to be stable in light therefore this preparation method was not used for the definitive test.
For the purpose of the definitive test the test material was dissolved directly in reconstituted water. An amount of test material (200 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 2 minutes and the volume adjusted to 2 litres to give the 100 mg/L test concentration. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.

- Test Water: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.
-- Reconstituted Water:
i) Stock Solutions
a) CaCl2 * 2H2O 11.76 g/L
b) MgSO4 * 7H2O 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCl 0.23 g/L
ii) Preparation
An aliquot (25 mL) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of < 5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3 .

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna
- Source: 1st instar, derived from in-house aboratory cultures.
- Feeding during test: none


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 21°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study. The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

-

Post exposure observation period:

-

Test conditions

Hardness:

250 mg/L as CaCO3 .

Test temperature:

20.9°C to 22.2°C. (Two temperatures in the test vessels were measured to be above the 21 ± 1°C range given in the protocol at 0 hours. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the test and that the deviation was only by 0.2°C.)

pH:

The reconstituted water had a pH of 7.8 ± 0.2. The pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. There were no treatment related differences for pH.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value. The test vessels were not aerated. The dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. There were no treatment related differences for oxygen concentration.

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC 50 values in terms of the nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: jars
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: 250 mL glass jars containing approximately 200 mL of test preparation
- Aeration: the test vessels were not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static)
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water:
i) Stock Solutions
a) CaCl 2 .2H 2 O 11.76 g/L
b) MgSO 4 .7H 2 O 4.93 g/L
c) NaHCO 3 2.59 g/L
d) KCl 0.23 g/L
ii) Preparation
An aliquot 25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3 .

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 1.0, 10 and 100 mg/L.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

Analysis of the test preparations at 0 and 48 hours (see Appendix 1) showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC 50 values in terms of the nominal test concentrations only.

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

Analysis of the test preparations at 0 and 48 hours (see Appendix 1) showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC 50 values in terms of the nominal test concentrations only.

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

Analysis of the test preparations at 0 and 48 hours (see Appendix 1) showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC 50 values in terms of the nominal test concentrations only.

Details on results:

- Mortality of control: no mortality observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No. Throughout the duration of the test the test preparations were observed to be clear, colourless solutions.

Reported statistics and error estimates:

-

Any other information on results incl. tables

Verification of Test Concentrations

Sample	Nominal Concentration (mg/l)	Concentration Found (mg/l)	Expressed as a Percent of the Nominal Concentration (%)
0 Hours	Control	<LOQ	-
"	100 R1 - R2	99.5	100
"	100 R3 - R4	99.5	99
48 Hours	Control	<LOQ	-
"	100 R1 - R2	97.3	97
"	100 R3 - R4	97.6	98

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a limit test with Daphnia magna, the test substance showed 48h-EC50 and 48h-NOEC to be >100 mg/L nominal (analytically verified).