Pyrimidine, 2-ethoxy-4,6-difluoro-

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:

17 December 1993 to 29 November 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-4,6-difluoropyrimidine

EC Number:

692-762-1

Cas Number:

166524-65-8

Molecular formula:

C6H6F2N2O

IUPAC Name:

2-ethoxy-4,6-difluoropyrimidine

Test material form:

other: liquid (unspecified)

Details on test material:

- Name of test material (as cited in study report): 2-ethoxy-4,6-difluoropyrimidine
- Appearance: clear liquid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Two initial stock solutions were prepared at concentrations of 0.40 and 0.20 mg/mL. Test solutions were prepared by serial dilution of the stock solutions. Acetone was used as a vehicle.

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 ± 193 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:

150 mg/L (as CaCO₃)

Test temperature:

201. - 21.6 °C

pH:

7.3 - 7.7

Dissolved oxygen:

8.4 - 8.8 mg/L (> 95 % saturation)

Nominal and measured concentrations:

Nominal treatment levels: 0 (water control), 0 (solvent control), 1.68, 2.81, 4.68, 7.80 and 13.00 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL borosilicate glass jars
- Type: 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
- No. of vessels per vehicle control (replicates): 4

DILUTION WATER
- Supplier: The City of Midland Water Treatment Plant
- Source: The upper Saginaw Bay of Lake Huron off Whitestone Point
- 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.
> Analysis of dilution water:
- Hardness (as CaCO₃): 150 mg/L
- Alkalinity (as CaCO₃): 47 mg/L
- pH: 7.7
- Conductivity: 390 µmhos/cm
- Residual chlorine: Not detected at greater than 20 µg/L
- Chloride: 74 000 ± 2000 µg/L
- Total organic carbon: 1565 µg/L
- Particulate matter (TSS): Not detected at greater than 1000 µg/L
- Metals: Aluminium 73 µg/L; Calcium 55 000 µg/L; Magnesium 8600 µg/L; Potassium 1100 µg/L; Sodium 4300 ± 300 µg/L.
- Organochlorine pesticides, chlorophenoxy herbicides, organophosphate insecticides and polychlorinated biphenyls: All below the 0.5 µg/L detection limits.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 2100 ± 152 lux

EFFECT PARAMETERS MEASURED
All daphnia were observed daily for mortality (no visible heartbeat or no response to gentle prodding) and sub-lethal effects (immobility or floating on the surface). Dead instars were not removed from the test vessels. Observations were performed at 0, 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.

TEST CONCENTRATIONS
- Range finding study: Yes.
Two range-finder studies were conducted. In the first study, there was an insufficient number of instars to adequately evaluate the toxicity of the test material. Therefore, a second range-finder study was conducted. The results of the second study indicated that the 48-hour LC50 value was less than 13.0 mg/L.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

3.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL 2.77 - 3.91 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and sub-lethal effects

Remarks on result:

other: 95 % CL 1.52 - 2.27 mg/L

Details on results:

The 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 intervals. This program had three methods available: probit analysis, moving average angle analysis and binomial probability/non-linear interpolation. The probit analysis and moving average methods calculate both the estimated EC50/LC50 value and its confidence interval. The binomial method calculates only the confidence interval, while a point estimate of the EC50/LC50 is obtained using non-linear interpolation, i.e., log transformation of the concentration and angle transformation of the number dead. The moving average method is less rigorous, statistically, than the probit; the binomial/non-linear interpolation method is less rigorous than the moving average. The order of preference used to select the method for reporting EC50/LC50 values is: probit analysis, moving average and binomial/non-linear interpolation. However, the appropriateness of a given method is determined in the program by the concentration-response data, e.g., the number of concentrations resulting in adverse effects between 0 and 100 percent.

Any other information on results incl. tables

Table 1: Concentration-Response Data

Nominal Conc. (mg/L)	Number Dead (%)		Number Affected (%)*
	24 hr	48 hr	24 hr	48 hr
0 (dilution water)	0 (0)	0 (0)	5 (20)	4 (16)
0 (solvent control)	0 (0)	0 (0)	0 (0)	0 (0)
1.68	0 (0)	3 (16)	6 (32)	7 (37)
2.81	0 (0)	5 (24)	0 (0)	17 (81)
4.68	0 (0)	15 (75)	2 (10)	20 (100)
7.80	0 (0)	21 (100)	0 (0)	21 (100)
13.00	0 (0)	19 (100)	1 (5)	19 (100)

*Effects were recorded as deaths, floating and immobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48 hour EC50 value was determined to be 1.94 mg/L with 95 % confidence limits of 1.52 - 2.27 mg/L. The 48 hour LC50 value was determined to be 3.31 mg/L with 95 % confidence limits of 2.27 - 3.91 mg/L. A No Observed Effect Concentration (NOEC) was not established.

Executive summary:

The acute toxicity of the test material to the water flea, Daphnia magna Straus, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OTS 797.1300 and OECD 202.

During the study, four replicate groups of five water fleas were exposed to nominal treatment levels of 1.68, 2.81, 4.68, 7.80 and 13.00 mg/L under static conditions for 48 hours. Four replicate groups of five water fleas were also used for laboratory water and solvent controls.

All daphnia were observed daily for mortality (no visible heartbeat or no response to gentle prodding) and sub-lethal effects (immobility or floating on the surface).

Under the conditions of the study the 48 hour EC50 value was determined to be 1.94 mg/L with 95 % confidence limits of 1.52 - 2.27 mg/L. The 48 hour LC50 value was determined to be 3.31 mg/L with 95 % confidence limits of 2.27 - 3.91 mg/L. A No Observed Effect Concentration (NOEC) was not established.