4(3H)-Pyrimidinone, 2-ethoxy-6-hydroxy-

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:

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:

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-6-hydroxy-3,4-dihydropyrimidin-4-one

EC Number:

612-185-0

Cas Number:

61636-08-6

Molecular formula:

C6H8 N2 O3

IUPAC Name:

2-ethoxy-6-hydroxy-3,4-dihydropyrimidin-4-one

Test material form:

other: solid (unspecified)

Details on test material:

- Appearance: white to light tan solid

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 7 mL of acetone, were added to 15 L of dilution water. No correction was made for the test material purity.
The test material had not completely dissolved in 30 minutes for all dose levels. After 24 hours no undissolved material remained at any dose level.

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): 35.6 mm (33 to 41 mm)
- Mean weight: 0.61 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

Hardness:

70 mg/L as CaCO₃

Test temperature:

12 ± 1 °C (ranged from 12.0 - 12.5 °C)

pH:

5.9 to 7.8

Dissolved oxygen:

>79 % of saturation (8.3 mg/L) at 12 °C (ranged from 8.3 - 8.9 mg/L)

Nominal and measured concentrations:

Nominal treatment levels: 0 (water control), 0 (acetone control), 4.95, 9.65, 19.5, 39.0, 79.5 and 159.5 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessels: 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: 0.41 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: ~1567 lux
- Test solutions: The 15 L of water in each of the treated tanks were dosed by the addition of 2.393, 2.38, 1.2, 1.179, 0.59, 0.592, 0.288, 0.302, 0.15, 0.14, 0.073, and 0.075 g of test material to give the two replicates for each nominal concentration.

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.:

> 159.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

159.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and sub-lethal effects

Details on results:

No fish exhibited adverse effects or died at any concentration level at any time during the test.

Reported statistics and error estimates:

A computer program was used to calculate the LC50 value. 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: Mortality and Effect Data

Nominal Concentration (mg/L)	Replicate Aquaria	Observation	Time period (hours)
			24	48	72	96
Water Control	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
Acetone Control	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
159.5	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
79.5	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
39.0	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
19.5	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
9.65	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0
4.95	A	Dead Affected	0 0	0 0	0 0	0 0
	B	Dead Affected	0 0	0 0	0 0	0 0

The number of fish per replicate was 10.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 96 h LC50 of the test material to rainbow trout was determined to be >159.5 mg/L and the NOEC determined as 159.5 mg/L, based on nominal concentrations.

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, fish were exposed to nominal treatment levels of 4.95, 9.65, 19.5, 39.0, 79.5 and 159.5 mg/L under static conditions for 96 hours. Groups of fish were also exposed to solvent (acetone) and laboratory water controls.

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).

No fish exhibited adverse effects or died at any concentration level at any time during the test.

Therefore, under the conditions of this study, the 96 h LC50 of the test material to rainbow trout was determined to be >159.5 mg/L and the NOEC determined as 159.5 mg/L, based on nominal concentrations.