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

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:

23 November 1993 to 1 August 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

Qualifier:

according to guideline

Guideline:

other: 40 CFR PART 797.1440 TOXIC SUBSTANCES CONTROL ACT

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
Appropriate amounts of test material, dissolved in 5 mL of acetone, were added to 15 L of dilution water. No correction was made for the test material purity.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Mean standard length (range): 34.3 mm (31 to 38 mm)
- Mean weight: 0.552 g
- Feeding during test: No. Additionally, fish were not fed in the 24 hours preceding the test.

REARING AND ACCLIMATION
- Acclimation period: At least 14 days
- Acclimation conditions: The test organism was obtained as eggs and milt. The eggs were fertilised then hatched, acclimated, and reared in a temperature-controlled environment having a 16 hour light/8 hour dark photoperiod with approximately 15 minute dusk and dawn transitions. Room illumination was provided by cool-white fluorescent lights activated by an automatic timer. Fish were acclimated to 12 ± 2 °C.
- Type and amount of food: Standard laboratory diet
- Feeding frequency: At least once daily
- Health during acclimation (any mortality observed): Mortality of the test lot was less than 0.1 % per day in the 14 days prior to testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Test temperature:

12.4 - 13.1 °C

pH:

7.4 - 7.8

Dissolved oxygen:

7.8 mg/L (> 75 % of saturation) at 12.5 °C

Nominal and measured concentrations:

Nominal treatment levels (first experiment): 0 (water control), 0 (acetone control), 20.5, 40.5, 81.5, 162, 325 and 537 mg/L.
Nominal treatment levels (second experiment): 0 (water control), 0 (acetone control), 1.70, 3.00, 5.20, 7.55 and 13.3 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 18 L borosilicate glass aquaria (~20 x 39.5 x 24.5 cm)
- Type (delete if not applicable): Closed. Aquaria were fitted with glass or plexiglass covers to retard evaporation and prevent fish from escaping.
- Fill volume: 15 L
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 0.37 g fish/L test solution

DILUTION WATER: softened Lake Huron water
- Supplier: The City of Midland Water Treatment Plant
- Source: The upper Saginaw Bay of Lake Huron off Whitestone Point
- Pre-treatment of water: The water was limed and flocculated with ferric chloride prior to arrival at the test facility. The water was subsequently sand-filtered, pH adjusted with CO₂, carbon-filtered, passed through 5 µm filters and UV irradiated in the laboratory before use.
- Water analysis: The water was monitored weekly for alkalinity, total chlorine, conductivity, hardness and pH. Analyses were conducted three times per year for total organic carbon, total suspended solids and selected inorganic and organic chemicals.
> Analysis of dilution water:
- Hardness (as CaCO₃): 70 mg/L
- Alkalinity (as CaCO₃): 49 mg/L
- pH: 7.8
- Conductivity: 250 µmhos/cm
- Chlorine: 4.0 ppb
- Total organic carbon: Not detected at greater than 1000 µg/L
- Metals: Aluminium 29 µg/L; Calcium 18 000 µg/L; Magnesium 7300 µg/L; Potassium 880 µg/L; Sodium 4400 ± 100 µg/L.
- Organochlorine pesticides, chlorophenoxy herbicides, organophosphate insecticides and polychlorinated biphenyls: All below the 0.5 µg/L detection limits.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark with 15 minute dawn and dusk transitions
- Light intensity: 1335 lux
- Test solutions: In the first set of exposures, the 15 L of water in each of the treated tanks were dosed by the addition of 0.316, 0.3, 0.599, 0.616, 1.222, 1.224, 2.423, 2.425, 4.882, 4.875, 8.064 and 8.047 g of test material to give the two replicates for each of the nominal concentrations 20.5, 40.5, 81.5, 162, 325 and 537 mg/L.
In the second set of exposures, the 15 L of water in each of the treated tanks were dosed by the addition of 0.024, 0.027, 0.047, 0.043, 0.077, 0.079, 0.114, 0.113, 0.199 and 0.198 g of test material to give the two replicates for each of the nominal concentrations 1.70, 3.00, 5.20, 7.55 and 13.3 mg/L.

EFFECT PARAMETERS MEASURED
The fish were observed daily for mortality (no response to gentle prodding and no opercular movement) and sub-lethal effects (e.g. swimming at the surface, complete or partial loss of body equilibrium, lethargy, erratic movement and exophthalmia). Dead fish, if any, were removed when observed.
All surviving test organisms were euthanised at test termination using tricaine methanesulfonate. Weight and standard length measurements were made on the laboratory water control fish at test termination.

WATER PARAMETERS MEASURED
Measurements of dissolved oxygen, pH and temperature were made at 1, 48 and 96 hours after initiation of testing in all test and control vessels with surviving fish.

TEST CONCENTRATIONS
- Justification for test concentrations: Test concentrations were based on preliminary estimates of test material toxicity which were obtained using the USEPA Structure Activity Relationship (SAR) computer program (Version 1.01, 1993).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 8.26 - 10.67 mg/L (95 % CL)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

Due to high mortality rates among fish exposed to the nominal concentrations of test material of greater than 20 mg/L in the first set of exposures (20.5, 40.5, 81.5, 162, 325 and 537 mg/L), a subsequent second series of nominal test material concentrations were investigated up to 13.3 mg/L (1.70, 3.00, 5.20, 7.55 and 13.3 mg/L).

In the second round of testing, one fish died following 96 hours of exposure to a nominal concentration of 5.20 mg/L; one fish died following 72 hours of exposure and a second fish died following 96 hours of exposure to a nominal concentration of 7.55 mg/L. Seventeen fish died following 72 hours of exposure to a nominal concentration of 13.3 mg/L; all fish exposed to 13.3 mg/L had died following 96 hours of exposure.
A complete set of test data for individual test vessels is provided in Tables 1 and 2.

Reported statistics and error estimates:

A computer program was used to calculate the LC50 value and corresponding 95 % confidence intervals. The program had three methods available: probit analysis, moving average angle analysis and binomial probability/non-linear interpolation. The probit analysis and moving average methods gave both the estimated LC50 value and its confidence interval. The binomial method gave only the confidence interval, while a point estimate of the LC50 was obtained using non-linear interpolation, i.e., log transformation of the concentration and angle transformation of the number dead.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1: Results from the First Series of Exposures

Nominal Conc. (mg/L)	Replicate Aquaria	Observation	Time Period† (hours)
			24	48	72
Water Control	A	Dead*	0	0	0
	A	Affected#	0	0	0
	B	Dead	0	0	0
	B	Affected	0	0	0
Solvent Control	A	Dead	0	0	0
	A	Affected	0	0	0
	B	Dead	0	0	0
	B	Affected	0	0	0
536	A	Dead	10	10	10
	A	Affected	10	10	10
538	B	Dead	10	10	10
	B	Affected	10	10	10
325	A	Dead	10	10	10
	A	Affected	10	10	10
325	B	Dead	10	10	10
	B	Affected	10	10	10
162	A	Dead	10	10	10
	A	Affected	10	10	10
162	B	Dead	10	10	10
	B	Affected	10	10	10
82	A	Dead	10	10	10
	A	Affected	10	10	10
81	B	Dead	10	10	10
	B	Affected	10	10	10
41	A	Dead	0	10	10
	A	Affected	10	10	10
40	B	Dead	0	10	10
	B	Affected	7	10	10
20	A	Dead	0	6	10
	A	Affected	1	10	10
21	B	Dead	0	4	10
	B	Affected	0	10	10

†All organisms in treatment tanks were dead at 72 hours; therefore controls were sacrificed at that time

*Number dead, n = 10

#Number affected (dead, loss of equilibrium, and/or lethargic), n = 10

Table 2: Results from the Second Series of Exposures

Nominal Conc. (mg/L)	Replicate Aquaria	Observation	Time Period (hours)
			24	48	72	96
Water Control	A	Dead*	0	0	0	0
	A	Affected#	0	0	0	0
	B	Dead	0	0	0	0
	B	Affected	0	0	0	0
Solvent Control	A	Dead	0	0	0	0
	A	Affected	0	0	0	0
	B	Dead	0	0	0	0
	B	Affected	0	0	0	0
13.2	A	Dead	0	0	8	10
	A	Affected	0	4	10	10
13.3	B	Dead	0	0	9	9
	B	Affected	0	4	10	10
7.5	A	Dead	0	0	1	1
	A	Affected	0	0	10	5
7.6	B	Dead	0	0	0	1
	B	Affected	0	0	0	3
5.3	A	Dead	0	0	0	0
	A	Affected	0	0	0	0
5.1	B	Dead	0	0	0	1
	B	Affected	0	0	0	1
2.9	A	Dead	0	0	0	0
	A	Affected	0	0	0	0
3.1	B	Dead	0	0	0	0
	B	Affected	0	0	0	0
1.8	A	Dead	0	0	0	0
	A	Affected	0	0	0	0
1.6	B	Dead	0	0	0	0
	B	Affected	0	0	0	0

*Number dead, n = 10

#Number affected (dead, loss of equilibrium, and/or lethargic), n = 10

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 96-hour LC50 of the test material, with rainbow trout, was determined to be 9.33 mg/L with lower and upper 95 % confidence limits of 8.26 and 10.67 mg/L, respectively. The No Observed Effect Concentration was determined to be 3.00 mg/L.

Executive summary:

The acute toxicity of the test material to Oncorhynchus mykiss (rainbow trout) was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline 40 CFR Part 797.1440 of the Toxic Substances Control Act.

During the study, duplicate groups of ten fish were exposed to nominal treatment levels of 1.70, 3.00, 5.20, 7.55, 13.3, 20.5, 40.5, 81.5, 162, 325 and 537 mg of test material per litre, in a static test, for 96 hours. Duplicate groups of ten fish were also exposed to solvent and laboratory water controls.

The test material had not completely dissolved at 30 minutes in dose levels greater than 81.5 mg/L. After 48 hours no undissolved test material remained for any dose level.

The fish were observed daily for mortality (no response to gentle prodding and no opercular movement) and sub-lethal effects (e.g. swimming at the surface, complete or partial loss of body equilibrium, lethargy, erratic movement and exophthalmia).

Under the conditions of the study, the 96-hour LC50 of the test material, with rainbow trout, was determined to be 9.33 mg/L with lower and upper 95 % confidence limits of 8.26 and 10.67 mg/L, respectively. The No Observed effect Concentration (NOEC) was determined to be 3.00 mg/L.