4,6-Dichloro-2-ethoxypyrimidine

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 November 1998 to 19 November 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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:

yes

Remarks:

Test solutions were not analytically verified.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Deviations:

yes

Remarks:

Test solutions were not analytically verified.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,6-Dichloro-2-ethoxypyrimidine

EC Number:

805-107-3

Cas Number:

40758-65-4

Molecular formula:

C6H6Cl2N2O

IUPAC Name:

4,6-Dichloro-2-ethoxypyrimidine

Test material form:

other: liquid

Details on test material:

- Physical state: liquid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solutions were prepared from a primary stock of test material that was formulated in N,N-dimethyl formamide (DMF) at a nominal concentration of 200 mg test material per mL DMF. Via serial dilution, stocks of 100, 50, 25, 12.5 and 6.25 mg test material per mL DMF were also prepared. From these stocks, bulk dosing solutions were prepared at nominal concentrations of 100, 50.0, 25.0, 12.5, 6.25 and 3.13 mg test material per litre of daphnid water. Daphnid dilution water and DMF control test vessels were also prepared.

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) 1000 - 2000 lux; 16 hour light / 8 hour dark photoperiod; temperature 20 ± 2 °C. Daphnids were fed a diet of Ankistrodesmus convolutus (algae) and YCT (yeast-ceraphyll trout) trout chow four 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, which have had at least four broods, were removed from the incubator. The instars were 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 were then placed in another stock tank that contained adjusted laboratory 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 repeated on the day the study was set to cull <24 hour old instars for use in the study.
- Feeding during test: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

162 mg/L (as CaCO3)

Test temperature:

19.3 - 20.9 °C

pH:

7.5 - 8.3

Dissolved oxygen:

8.3 - 8.7 mg/L

Nominal and measured concentrations:

0 (water control), 0 (DMF control), 3.13, 6.25, 12.5, 25.0, 50.0, 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type (delete if not applicable): closed (glass jars were covered to reduce evaporation)
- Fill volume: 200 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per water control (replicates): 2
- No. of vessels per solvent control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point by the City of Midland Water Treatment Plant.
- Treatment of dilution water: the Lake Huron water was limed and flocculated with ferric chloride. Before use in the study the water was sand-filtered, pH adjusted with gaseous CO2, carbon filtered and UV-irradiated. The daphnid water was adjusted to a hardness of about 170 mg/L (as CaCO3) before autoclaving. After adjusting the hardness, the water was autoclaved at 250 °C and 18 psi for 30 minutes, cooled and aerated for approximately 24 hours prior to use.
- Total organic carbon: 5798 ng/mL (laboratory dilution water); 4756 ng/mL (daphnid water)
- Total suspended solids: not detected (detection limit - 1000 ng/mL)
- Alkalinity: 30 mg/L (as CaCO3)
- Conductivity: 373 µmhos/cm
- Intervals of water quality measurement: laboratory and daphnid waters were monitored weekly for pH, alkalinity and conductivity and hardness; laboratory and daphnid waters were monitored twice yearly for total organic carbon, total suspended solids, selected inorganics and organic compounds.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 1833 ± 350 lux

EFFECT PARAMETERS MEASURED
Daphnids were observed for mortality (no visible heartbeat or response to gentle prodding) and immobility at 24 and 48 hours.

PHYSICAL MEASUREMENTS
Measurements of dissolved oxygen, pH and temperature were taken from the bulk solutions on day 0 and from each test vessel at 48 hours. Temperature was also continuously monitored within the incubator.

TEST CONCENTRATIONS
- Range finding study: yes, 48 hour study.
- Test concentrations: 0.001 to 10.0 mg/L
- Results used to determine the conditions for the definitive study: yes, no mortality or sublethal effects were observed at any of the dose levels tested.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

40.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 35.9 - 46.0 mg/L (95 % CL)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

22.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and immobility

Remarks on result:

other: 19.4 - 26.1 mg/L (95 % CL)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and immobility

Details on results:

Mortality and mobility 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 reported LC50/EC50 values were calculated via trimmed Spearman-Karber.

Any other information on results incl. tables

Table 1: Summary of Concentration-Response Data

Nominal Conc. (mg/L)	Observed Mortality		Observed Mortality and Immobility
	24 hours	48 hours	24 hours	48 hours
0 (water control)	0	0	0	0
0 (DMF control)	0	0	0	0
3.13	0	0	0	0
6.25	0	0	0	0
12.5	0	0	0	0
25.0	0	0	1	13
50.0	1	16	16	20
100	12	20	20	20
LC50 (mg/L)	88.2	40.6	-	-
95 % CL (mg/L)	70.3 - 111	35.9 - 46.0	-	-
EC50 (mg/L)	-	-	39.2	22.5
95 % CL (mg/L)	-	-	34.1 - 45.2	19.4 - 26.1

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 40.6 mg/L with 95 % confidence limits of 35.9 - 46.0 mg/L and the 48 hour EC50 value was determined to be 22.5 mg/L with 95 % confidence limits of 19.4 – 26.1 mg/L. The No Observed Effect Concentration (NOEC) was 12.5 mg/L.

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 guidelines EPA OTS 797.1300 and EPA OPP 72-2.

During the study, groups of 20 Daphnia were exposed to nominal treatment levels of 0 (laboratory water control), 0 (solvent control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L of test material. Daphnids were observed daily for mortality (no visible heartbeat or response to gentle prodding) and immobility.

Under the conditions of the study, the 48 hour LC50 value was determined to be 40.6 mg/L with 95 % confidence limits of 35.9 - 46.0 mg/L and the 48 hour EC50 value was determined to be 22.5 mg/L with 95 % confidence limits of 19.4 – 26.1 mg/L. The No Observed Effect Concentration (NOEC) was 12.5 mg/L.