[1,2,4]Triazolo[1,5-c]pyrimidine, 2,2'-dithiobis[5-ethoxy-7-fluoro-

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:

28 December 1993 to 30 December 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. No analytical determination of test substance concentration.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

EC Number:

695-187-4

Cas Number:

166524-75-0

Molecular formula:

C14H12F2N8O2S2

IUPAC Name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

Test material form:

other: solid (unspecified)

Details on test material:

- Appearance: white to light beige solid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Two initial stock solutions were prepared at concentrations of 0.16 and 0.016 mg/mL. Test solutions were prepared by serial dilution of the stock solutions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: laboratory reared culture
- Maintenance conditions: adult Daphnia were maintained in 2 L stock tanks.
- Rearing conditions: illumination (cool-white fluorescent) 2122 ± 199 lux; 16 hour light / 8 hour dark photoperiod; temperature 20 ± 2 °C. Daphnids were fed an algal diet of Selenastrum capricornutum three times weekly.
- Age at study initiation: <24 hours. The day before instars were needed for testing, stock tanks with daphnids greater than 14 days old were removed from the incubator and the instars separated from adults by gently lifting the screened insert from the 2-L stock tank, releasing instars through the nylon mesh screen while retaining the adult daphnids. The screened insert with adult daphnids was then placed in another stock tank that contained daphnid dilution water. The original solution with instars was poured through a metal sieve into another stock tank. The instars collected on the sieve were discarded and the original solution was poured back into the initial stock tank and the corresponding screened insert holding adult daphnids was put back in place. This procedure was then repeated to obtain <24 hour old instars for the test.
- Feeding during test: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

168 mg/L (as CaCO3)

Test temperature:

19.1 - 20.9 °C

pH:

7.7 - 8.0

Dissolved oxygen:

7.0 - 8.5 mg/L (> 79 % saturation)

Nominal and measured concentrations:

0 (control), 0.3, 0.5, 0.8, 1.2, 2.1, 3.5, 5.8, 9.6, 16.0, 26.7 and 44.4 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL borosilicate glass vessels
- Type (delete if not applicable): closed (test vessels were covered with watch glasses to reduce evaporation)
- Fill volume: 150 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point via the City of Midland Water Treatment Plant.
- Treatment of dilution water: the Lake Huron water was adjusted to a hardness of about 170 mg/L as CaCO3. The water was then autoclaved at 121 °C and 18 psi for 30 minutes, cooled, and aerated before use.
- Analysis of dilution water: the water was analysed weekly for hardness, alkalinity, conductivity and pH and three times a year for selected inorganics, total suspended solids, total organic carbon and selected organic compounds.
- Total organic carbon: 1330 µg/L
- Total suspended solids: 3200 µg/L
- Alkalinity: 48 mg/L (as CaCO3)
- Conductivity: 500 µmhos/cm
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 2033 ± 252 lux

EFFECT PARAMETERS MEASURED
All Daphnia were observed daily for mortality (no visible heartbeat or no response to gentle prodding) and sublethal effects (immobility or floating on the surface). Observations were performed at 3, 6, 12, 24 and 48 hours.

PHYSICAL MEASUREMENTS
Measurements of dissolved oxygen, pH and temperature were taken daily in each vessel from each test concentration and control. The temperature of the incubator was also continuously recorded.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

21.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 1.6 - 37.1 mg/L (95 % CL)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and mobility

Remarks on result:

other: 4.2 - 8.1 mg/L (95 % CL)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and mobility

Details on results:

Incomplete mixing (settling out of solution) was observed at the three highest exposure concentrations (16.0, 26.7 and 44.4 mg/L).
Concentration mortality and effect data are presented in Table 1.

Reported statistics and error estimates:

A computer program was used to calculate the EC50 and LC50 values and corresponding 95 % confidence limits. The probit analysis and moving average methods were used to calculate both the estimated EC50/LC50 values and confidence limits.

Any other information on results incl. tables

Table 1: Concentration-Response Data

Nominal Conc. (mg/L)	Number dead		Number Affected*
	24 hours	48 hours	24 hours	48 hours
0 (control)	0	0	0	0
0.3	0	0	0	0
0.5	0	0	0	1
0.8	0	0	0	1
1.2	0	0	0	5
2.1	0	0	0	5
3.5	0	5	0	11
5.8	0	7	0	12
9.6	0	8	0	11
16.0	0	10	0	12
26.7	0	8	0	17
44.4	0	12	0	18

*Effects were recorded as deaths and immobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48 hour LC50 value was determined to be 21.5 mg/L with 95 % confidence limits of 14.6 - 37.1 mg/L and the 48 hour EC50 value was determined to be 5.7 mg/L with 95 % confidence limits of 4.2 - 8.1 mg/L. The No Observed Effect Concentration (NOEC) was 0.3 mg/L. Results are based on nominal test concentrations.

Executive summary:

The acute toxicity of the test material to the aquatic invertebrate Daphnia magna was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OTS 797.1300.

During the study, groups of 20 Daphnia were exposed under static conditions for 48 hours to nominal treatment levels of 0 (laboratory water control), 0.3, 0.5, 0.8, 1.2, 2.1, 3.5, 5.8, 9.6, 16.0, 26.7 and 44.4 mg/L of test material. Incomplete mixing (settling out of solution) was observed at the three highest exposure concentrations (16.0, 26.7 and 44.45 mg/L).

Under the conditions of the study, the 48 hour LC50 value was determined to be 21.5 mg/L with 95 % confidence limits of 14.6 - 37.1 mg/L and the 48 hour EC50 value was determined to be 5.7 mg/L with 95 % confidence limits of 4.2 - 8.1 mg/L. The No Observed Effect Concentration (NOEC) was 0.3 mg/L. Results are based on nominal test concentrations.