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

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 9 May 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:

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:

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:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Three initial stock solutions were prepared at concentrations of 0.48, 0.048 and 0.0048 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) 2067 ± 187 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:

20 ± 1 °C (ranged from 19.0 - 20.8 °C)

pH:

6.4 to 8.0

Dissolved oxygen:

7.1 to 8.7 mg/L (>81 % saturation)

Nominal and measured concentrations:

Nominal treatment levels: 0 (dilution water control), 0.3, 0.5, 0.8, 1.3, 2.2, 3.7, 6.2, 10.4, 17.3, 28.8, 48.0, 80.0 and 133.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL borosilicate glass vessels
- 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

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): 168 mg/L
- Alkalinity (as CaCO₃): 48 mg/L
- pH: 7.9
- Conductivity: 500 µmhos/cm
- Residual chlorine: Not detected at greater than 20 µg/L
- Chloride: 86 000 ± 3000 µg/L
- Total organic carbon: 1330 µg/L
- Particulate matter (TSS): 3200 µg/L
- Metals: Aluminium 89 µg/L; Calcium 62 000 µg/L; Magnesium 8000 µg/L; Nickel 11 µg/L; Potassium 1100 µg/L; Sodium 5300 ± 200 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: 1967 ± 306 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.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 133.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 133.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and sub-lethal effects

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 Concentrations (mg/L)	Number Dead (%)		Number Affected* (%)
	24 Hours	48 Hours	24 Hours	48 Hours
Dilution Water Control	0 (0)	0 (0)	0 (0)	0 (0)
133.3	0 (0)	0 (0)	0 (0)	1 (5)
80.0	0 (0)	1 (5)	0 (0)	1 (5)
48.0	0 (0)	0 (0)	0 (0)	0 (0)
28.8	0 (0)	0 (0)	0 (0)	0 (0)
17.3	0 (0)	0 (0)	0 (0)	0 (0)
10.4	0 (0)	0 (0)	0 (0)	0 (0)
6.2	0 (0)	0 (0)	0 (0)	0 (0)
3.7	0 (0)	0 (0)	0 (0)	0 (0)
2.2	0 (0)	0 (0)	0 (0)	0 (0)
1.3	0 (0)	0 (0)	0 (0)	0 (0)
0.8	0 (0)	0 (0)	0 (0)	0 (0)
0.5	0 (0)	0 (0)	0 (0)	0 (0)
0.3	0 (0)	0 (0)	0 (0)	0 (0)

*Effects were recorded as deaths and immobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48 h LC50 and EC50 of the test material to Daphnia magna were determined to be > 133.3 mg/L, based on nominal concentrations.

Executive summary:

The acute toxicity of the test material to Daphnia magna STRAUS was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline 40 CFR Part 797.1300 of the Toxic Substances Control Act.

During the study, daphnids were exposed to nominal treatment levels of 0.3, 0.5, 0.8, 1.3, 2.2, 3.7, 6.2, 10.4, 17.3, 28.8, 48.0, 80.0 and 133.3 mg/L under static conditions for 48 hours. Daphnids were also exposed to laboratory water 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 this study, the 48 h LC50 and EC50 of the test material to Daphnia magna were determined to be >133.3 mg/L, based on nominal concentrations.