4,6-Dichloro-2-ethoxypyrimidine

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:

16 November 1998 to 20 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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

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 797.1400 (Fish Acute Toxicity Test)

Deviations:

yes

Remarks:

Test solutions were not analytically verified.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish 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
Test solutions of 15, 7.5, 3.75, 1.88, 0.94 and 0.47 mg/L were prepared by serial dilution of a concentrated stock solution (105 mg/mL) made in N,N'dimethyl formamide (DMF)

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Species: Oncorhynchus mykiss Walbaum
- Common name: rainbow trout
- Holding period: upon arrival, the juveniles were visually inspected and then placed in a 230 L holding tank. All fish were held on a 16 hour light / 8 hour dark transitional photoperiod and observed for at least 14 days before testing. During holding they received a standard diet at least once daily. The test lot used for the definitive study was acclimated to 12 ± 2 °C and held without food for at least 48 hours before testing.
- Health during acclimation (any mortality observed): mortality was less than 1 % of the population in the 48 hours before testing.
- Length at study initiation: the length of the largest fish was no more than twice the length of the shortest fish.
- Weight at study initiation: fish of approximately 0.5 g were used as test organisms.
- Feeding during test: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

58 mg/L (as CaCO3)

Test temperature:

11.4 - 12.5 °C

pH:

6.8 - 7.9

Dissolved oxygen:

4.8 - 9.9 mg/L (46 - 95 % saturation)

Dissolved oxygen levels were greater than 60 % saturation from 0 - 48 hours (with the exception of one 15.0 mg/L dose level test vessel in which mortality and decomposition was occurring) and 40 % of saturation after 48 hours; aeration was not required to maintain the required dissolved oxygen levels.

Nominal and measured concentrations:

0 (water control), 0 (DMF control), 0.47, 0.94, 1.88, 3.75, 7.5 and 15.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 4 L Pyrex borosilicate glass beakers
- Type (delete if not applicable): closed (each beaker was loosely covered with a glass plate)
- Fill volume: 3.5 L
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.835 g fish/L of test solution

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Huron water, obtained from the upper Saginaw Bay off Whitestone Point, was supplied by the City of Midland Water Treatment Plant. The water is limed and flocculated with ferric chloride.
- Preparation of dilution water: before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO2, carbon filtered and UV-irradiated.
- Total organic carbon: 5798 ng/mL (laboratory dilution water)
- Total suspended solids: not detected (detection limit = 1000 ng/mL)
- Chlorine: 11 ppb
- Alkalinity: 26 mg/L (as CaCO3)
- Conductivity: 139.3 µmho/cm
- Intervals of water quality measurement: Laboratory water was monitored weekly for pH, alkalinity, conductivity and hardness. Laboratory water was 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, provided by cool-white fluorescent lighting and an automatic timer

EFFECT PARAMETERS MEASURED
The fish were observed every 24 hours of exposure for mortality (no response to touching of the caudal peduncle and no opercular movement) and sublethal effects. Dead individuals were removed. Any behavioural effects were noted and reported, e.g. swimming at the surface, complete or partial loss of body equilibrium, lethargy, hyperactivity and erratic movement. Any gross pathological conditions were also noted and reported; e.g. exophthalmia, ascites, haemorrhage, excess mucus, sloughing of epidermis and melanosis.
At the end of the study, terminal body weight (nearest 1 mg) and length (nearest 1 mm) measurements were recorded from the control group and the highest surviving concentration as representative of the test population. All fish were euthanised with tricaine methanesulfonate prior to the length and weight measurements.

WATER PARAMETERS MEASURED
Measurements of dissolved oxygen, pH and temperature were made at 0, 48 and 96 hours after initiation of testing in all test and control vessels.

TEST CONCENTRATIONS
- Range finding study: yes.
- Test concentrations: 0.001 - 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 8.7 - 11.3 mg/L (95 % CL)

Details on results:

After 96 hours of exposure, there was 100 % mortality at the 15.0 mg/L concentration level. 10 % mortality was observed at the 7.5 mg/L concentration.
Melanosis was noted at concentrations down to 7.5 mg/L. Mortality and sublethal effects were absent in the laboratory water of DMF controls. See table 1 for further information.

Reported statistics and error estimates:

A computer program was used to calculate the LC50 vales and corresponding 95 % confidence limits.

Any other information on results incl. tables

Sublethal observations / clinical signs:

The mean length and weight for the control fish were 36 mm and 0.675 g, respectively and for the highest surviving concentration (3.75 mg/L) fish 32 mm and 0.495 g, respectively. The biological loading rate was 0.835 g fish/L of test solution.

Table 1: Summary of Survival Data

Nominal Conc. (mg/L)	Replicate	Observed Mortality
		24 hours	48 hours	72 hours	96 hours
0 (water control)	A	0	0	0	0
	B	0	0	0	0
0 (DMF control)	A	0	0	0	0
	B	0	0	0	0
0.47	A	0	0	0	0
	B	0	0	0	0
0.94	A	0	0	0	0
	B	0	0	0	0
1.88	A	0	0	0	0
	B	0	0	0	0
3.75	A	0	0	0	0
	B	0	0	0	0
7.5	A	0	0	0	0
	B	0	0	0	1
15.0	A	0	3	5	5
	B	0	1	5	5
LC50 (mg/L)		> 15.0	> 15.0	10.6	9.9
95 % CL (mg/L)		NA	NA	NA	8.7 - 11.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 96 hour LC50 was determined to be 9.9 mg/L, with 95 % confidence limits of 8.7 - 11.3 mg/L.

Executive summary:

The acute toxicity of the test material to Oncorhynchus mykiss was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OTS 797.1400 and EPA OPP 72-1.

During the study, groups of 10 rainbow trout were exposed to nominal concentrations of test material of 0 (laboratory water and solvent controls), 0.47, 0.94, 1.88, 3.75, 7.5 and 15.0 mg/L. Fish were exposed for 96 hours under static conditions. Mortality and sublethal effects were observed daily.

No mortality occurred at concentrations up to 3.75 mg/L. One fish exposed to 7.5 mg/L test material died on the fourth day of exposure. At the highest concentration (15.0 mg/L) 4 fish died on the second day of exposure; all fish had died by the third day of exposure. Melanosis was noted at concentrations down to 7.5 mg/L. Mortality and sublethal effects were absent in the laboratory water and solvent controls.

Under the conditions of the study, the 96 hour LC50 was determined to be 9.9 mg/L, with 95 % confidence limits of 8.7 - 11.3 mg/L.