4,6-Dichloro-2-ethoxypyrimidine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 1998 to 19 December 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:

other: EPA OPP 123-2

Deviations:

yes

Remarks:

Test solutions were not analytically verified.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)

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
An initial 100 mg/L primary stock solution was prepared by weighing out approximately 117.8 mg of the test material and dissolving it in 1.2 mL of N,N-dimethyl formamide (DMF).
Serial dilutions of the 100 mg/mL stock were prepared in DMF and a 50 µL aliquot of each of the concentration stocks was diluted with algal assay medium (AAM) to the mark in a 500 mL volumetric flask.
Control AAM and control medium containing 100 µL DMF/L AAM carrier were also prepared.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Species: Selenastrum capricornutum Printz
- Strain: 1648
- Age of inoculum (at test initiation): 6 days
- Maintenance of algae: stock cultures of algae were maintained aseptically by weekly transfer into sterile algal assay medium (AAM)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Test temperature:

24.2 - 25.4 °C

Nominal and measured concentrations:

0 (medium control), 0 (solvent control), 0.31, 0.63, 1.3, 2.5, 5 and 10 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilised 250 mL borosilicate Erlenmeyer flasks
- Type (delete if not applicable): closed (the flasks had Shimadzu closures)
- Fill volume: 100 mL
- Initial cell density: 11 000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per medium control (replicates): 3
- No. of vessels per solvent control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (Algal Assay Medium) The culture medium used for the definitive test was the same as that used to maintain the stock culture except that the medium used in the toxicity tests did not contain the chelant, Na2EDTA.2H2O
- Preparation of the Algal Assay Medium:
Each stock solution was filtered through a 0.45 µm membrane filter, then stored at 4 °C; the solutions were brought up to room temperature before using. 1 mL of each stock solution A, B1, B2, B3 and D, plus 10 mL of C were added to 900 mL of dilution water. The mixture was then made up to 1 L with additional water.
A: 12.75 g NaNO3, 6.08 g MgCl2.6H2O and 2.20 g CaCl2.2H2O was added to 500 mL dilution water
B1: 7.35 g MgSO4.7H2O dissolved in 500 mL dilution water
B2: 7.5 g NaHCO3 dissolved in 500 mL dilution water
B3: 0.522 g K2PO4 dissolved in 500 mL dilution water
C: 5 mL of number 1 (below) and 5 mL of number 3 (below) diluted to 500 mL with sterile dilution water.
1: 1.86 g H3BO3, 4.17 g MnCl2.4H2O, 0.0327 g ZnCl2 and 0.0726 g NaMoO4.2H2O dissolved in 1000 mL dilution water
2: 2.86 g CoCl2.6H2O and 0.22 g CuCl2.2H2O dissolved in 1000 mL dilution water
3: 2.5 mL of number 2 was made up to 500 mL
D: 0.30 g Na2EDTA.2H2O and 0.16 g FeCl3.6H2O dissolved in 1000 mL dilution water
Finally, the pH was adjusted to 7.0 to 7.5.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 3500 - 5250 lux

EFFECT PARAMETERS MEASURED
The algal densities of the inoculum and test cultures were determined by electron particle counting using a Coulter Multisizer. Total cell counts were determined on day 0 and after approximately 96 hours of exposure. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.7 µm to a higher threshold equivalent spherical diameter of approximately 8.6 µm. A blank correction for the algal assay medium used in preparing the dose solutions was also compensated for in the calculations.

PHYSICAL ANALYSIS
The incubator temperature was continuously monitored with a temperature probe which was placed in a representative vessel within the incubator. The light intensity was monitored daily at positions corresponding to the test flasks in the incubator.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: test concentrations were set in a geometric series using a progression factor of approximately 2.0
- Range finding study: yes
- Range finding study concentrations: 0.01, 0.1, 1 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes. The 4 day EC50 value in the range-finding study was greater than 1 mg/L and less than 10 mg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.62 - 6.63 mg/L (95 % CL)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects of the test material on algal growth, relative to the controls, ranged from 13.9 % inhibition of growth at 0.31 mg/L, to 81.9 % inhibition of growth at 10 mg/L by day 4.

The 96 hour EbC50 value for inhibition of growth was 1.98 mg/L with 95 % confidence limits of 0.68 - 5.75 mg/L.

Reported statistics and error estimates:

The EC50 for algal growth was determined by a least squares linear regression of algal cell counts against the common log of the concentrations on day 4. The EbC50 for inhibition of growth was calculated by the regression of the differences in area under the growth curves for each dose group compared to the control against the log of the concentrations for day 4. The No Observed Effect Concentration (NOEC) for algal growth was calculated using the analysis of variance and Dunnett's t test.

Any other information on results incl. tables

Table 1: Mean Values for Total Cell Count/mL

Nominal Conc. (mg/L)	Mean Total Cell Count/mL
	Day 0	Day 4
medium	12270	1237727
	13076	1255756
	11502	1110468
DMF	10696	946396
	11106	1115385
	11234	1143698
0.31	11144	994530
	10396	922399
	10922	1013174
0.63	11051	763782
	10541	876339
	11067	851225
1.3	11321	714943
	12751	669035
	11032	280775
2.5	10526	692344
	11105	568756
	10363	615408
5	10233	352460
	10833	331939
	10568	451382
10	11092	219184
	10659	156179
	9962	240837

Table 2: Mean Values for Inhibition of Growth

Nominal Conc. (mg/L)	Day 0	Day 4	% Inhibition (Day 4)
media	12283	1201317	-
DMF	11012	1068493	-
0.31	10820	976701	13.9
0.63	10886	830449*	26.8
1.3	11701	554918*	51.1
2.5	10665	625503*	44.9
5	10545	378594*	66.6
10	10571	205400*	81.9

* significant difference from the combined controls; p ≤ 0.05 one tailed Dunnett's t-test on raw data

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96 hour EbC50 for inhibition of growth was 1.98 mg/L. The 96 hour EC50 for algal cell growth was 2.03 mg/L and the No Observed Effect Concentration (algal cell growth) was 0.31 mg/L.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 123-2 and EPA OTS 797.1050.

During the study, the freshwater green alga Selenastrum capricornutum Printz was exposed to the test material at concentrations of 0 (medium control), 0 (solvent control), 0.31, 0.63, 1.3, 2.5, 5 and 10 mg/L for 96 hours. Three replicates were set in each treatment group, each with an initial cell density of 11 000 cells/mL. Effects of the test material on algal cell growth, relative to controls, ranged from 13.9 % inhibition of growth at 0.31 mg/L, to 81.9 % inhibition of growth at 10 mg/L by day 4. The EC50 values, together with the NOEC, were based on nominal concentrations.

Under the conditions of the study the 96 hour EbC50 for inhibition of growth was 1.98 mg/L (0.68 - 5.75 mg/L 95 % confidence limits). The 96 hour EC50 for algal cell growth was 2.03 mg/L (0.62 - 6.63 mg/L 95 % confidence limits) and the No Observed Effect Concentration (algal cell growth) was 0.31 mg/L.