Pyrimidine, 2-ethoxy-4,6-difluoro-

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:

25 October 1993 to 15 July 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Toxic Substances Control Act Test Guidelines

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-4,6-difluoropyrimidine

EC Number:

692-762-1

Cas Number:

166524-65-8

Molecular formula:

C6H6F2N2O

IUPAC Name:

2-ethoxy-4,6-difluoropyrimidine

Test material form:

other: liquid (unspecified)

Details on test material:

- Name of test material (as cited in study report): 2-ethoxy-4,6-difluoropyrimidine
- Appearance: clear liquid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test material was used as received; it was added directly to the test medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 1648

CULTURE CONDITIONS
Stock cultures of this organism were maintained axenically by weekly transfer into sterile algal assay medium (AAM).
- Temperature: 25 ± 2 °C
- Light: 4300 ± 650 lux
- Photoperiod: Continuous
- Medium: as for the AAM Bottle Test (EPA-600/9-78-018)
- pH: 7.0 to 7.5
- Septic conditions: Axenic
- Culture vessel: 500 mL Erlenmeyer flask
- Habitat maintenance: Inoculation: 4 to 5 days; Culture chamber: Incubator; Amount of transfer: 20 000 cells/mL; Culture agitation: ~100 rpm; Acclimation: 4 weeks.
- Health criteria: Adequate growth: 1 x 10⁶ cells/mL in 5 days

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

5 d

Test conditions

Test temperature:

24.2 - 24.6 °C

pH:

7.3 - 7.6 (without algae); 7.2 - 8.9 (with algae)

Nominal and measured concentrations:

Nominal treatment levels: 0 (medium control), 1.88, 3.75, 7.5, 15, 30 and 60 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised 250 mL borosilicate Erlenmeyer flasks.
- Type: Closed with Shimadzu closures.
- Fill volume: 100 mL (algal test medium)
- Aeration: Flasks were continuously agitated at approximately 100 rpm.
- Initial cell density: Approximately 10 000 cells/mL. The stock culture was used to prepare an algal inoculum by diluting with test medium, such that a 1 mL aliquot of the inoculum would provide the appropriate cell density.
- No. of vessels per concentration (replicates): Three. A counting blank was included with each test concentration (contained growth medium and the appropriate amount of test material but no algae).
- No. of vessels per control (replicates): Six

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, with the exception that the medium used in the toxicity tests did not contain the chelant Na₂EDTA.2H₂O. the medium was stored in the dark at approximately 4 °C.
- Preparation of the Algal Assay Medium:
Add one mL of each stock solution A, B1, B2, B3 and D, plus 10 mL of C to 900 mL of dilution water and make up to one litre with additional water. 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.
A: 12.75 g NaNO₃, 6.08 g MgCl₂.6H₂O and 2.20 g CaCl₂.2H₂O dissolved in 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 0.022 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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 4300 ± 1100 lux

EFFECT PARAMETERS MEASURED
The algal cell densities of the inoculum and test cultures were determined by electron particle counting using a Coulter Multisizer. Total cell counts were determined at approximately 24, 48, 72, 96 and 120 hours. Cells were cumulatively counted from a lower threshold equivalent spherical diameter of approximately 2.7 µm to a higher threshold equivalent spherical diameter of approximately 8.6 µm. The background particle count (growth medium and test material) was concurrently subtracted from that of the sample dilution (growth medium, test material and algae) to obtain cell density. A blank correction for the isotonic saline solution used in preparing the dilutions was also compensated for in the calculations.

PHYSICAL ANALYSIS
Extra flasks without algae were added to the test for each nominal concentration to monitor pH at the initiation of the test and every day thereafter. At the end of the test a final pH was taken in a replicate test flask at each test concentration with algae. The temperature of a representative test solution was continuously monitored during the test. The light intensity was checked on a daily basis with readings taken corresponding to the positions of the test flasks in the incubator.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Test concentrations were set in a geometric series with a progression factor of 2.0.
- Range finding study: Yes
- Range finding test concentrations: 0.1, 1.0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes; the results indicated an inhibition of cell growth through 100 mg/L and essentially no effect at 10 mg/L in comparison to the controls.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 4.3 - 71.5 mg/L (95 % CL)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 13.9 - 50.9 mg/L (95 % CL)

Effect concentrations 4

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

See Tables 1, 2 and 3.

Reported statistics and error estimates:

The results are expressed in terms of algal growth (total cell count/mL) and inhibition (percent). The EC50 values were determined for both endpoints. The EC10, EC50, and EC90 values for algal growth were numerically derived based on calculated regression equations for each 24 hour period. The no observed effect concentrations (NOECs) given for algal growth were calculated using analysis of variance and the Dunnett's test. The percent inhibition is a graphic interpretation of the data and the EC values were calculated by regression based on the comparison of the areas under the growth curves between the control group and each exposure concentration.

Any other information on results incl. tables

Table 1: Mean Values for Growth for Each Replicate Test Flask

Nominal Conc. (mg/L)	Day 1	Day 2	Day 3	Day 4	Day 5
60	8800	12720	15333	43853	109600
	9140	9827	9893	32440	88907
	16147	11147	7933	42493	125773
30	8953	23000	83707	178733	690000
	9773	13280	32493	107200	425333
	20993	17107	33867	133200	487933
15	14860	32813	66600	234933	940933
	10440	47667	98533	299600	1175600
	9960	35600	113267	336667	1100800
7.5	10107	44760	101733	418800	1267200
	10053	34453	86600	352667	1221733
	11447	33347	125933	461200	1331733
3.75	32480	31227	143067	518800	1545867
	11787	23067	118467	452267	1369733
	11793	44840	126267	420933	1249867
1.8	12500	43053	176200	533467	1625200
	11453	40640	94400	370533	1036933
	11633	38013	157533	456667	1376533
0 (control)	12600	43240	164067	606800	1608933
	11513	32107	140533	487200	1265467
	11973	29933	127200	432200	1190933
	13547	40013	141800	492133	1449867
	12907	32693	87667	264000	878267
	20433	39107	173533	541733	1457867

Table 2: Mean Five Day Test Results Showing the NOECs Using the One-tailed Dunnett’s Test

Nominal Concentration (mg/L)	Statistics	Day 1	Day 2	Day 3	Day 4	Day 5
60	Mean SD CV	11362 4146.9 36.5	11231* 1448.5 12.9	11053* 3834.0 34.7	39596* 6234.1 15.7	108093* 18479.5 17.1
30	Mean SD CV	13240 6727.1 50.8	17796* 4896.5 27.5	50022* 29179.7 58.3	139711* 36208.4 25.9	534422* 138322.2 25.9
15	Mean SD CV	11753 2701.1 23.0	38693 7895.1 20.4	92800 23855.8 25.7	290400* 51486.9 17.7	1072444 119875.5 11.2
7.5	Mean SD CV	10536 789.5 7.5	37520 6294.4 16.8	104756 19840.1 18.9	410889 54697.4 13.3	1273556 55274.7 4.3
3.75	Mean SD CV	18687 11945.4 63.9	33044 10999.9 33.3	129267 12571.4 9.7	464000 49977.2 10.8	1388489 148888.6 10.7
1.88	Mean SD CV	11862 559.6 4.7	40569 2520.8 6.2	142711 42867.0 30.0	453556 81511.2 18.0	1346222 295302.4 21.9
Control	Mean SD CV	13829 3312.5 24.0	36182 5308.0 14.7	139133 30349.1 21.8	470678 117053.6 24.9	1308556 258197.4 19.7

SD = Standard deviation

CV = Coefficient of variation

*Comparisons significant by Dunnett’s one-tailed test at the 0.05 level (i.e. p≤0.05)

Table 3: Percentage Inhibition of Growth in Comparison to the Control at Each 24 Hour Interval

Nominal Conc. (mg/L)	Day 1	Day 2	Day 3	Day 4	Day 5
60	64.4	88.3	96.7	95.3	93.5
30	15.4	57.8	67.2	70.2	65.1
15	54.2	4.8	24.0	34.9	27.1
7.5	86.0	15.5	20.2	17.0	9.0
3.75	-126.9	-19.4	3.4	2.9	-2.0
1.88	51.4	-1.3	-4.5	0.7	-0.6
0 (control)	0.0	0.0	0.0	0.0	0.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 and 120 hour EC50 values (and their 95 % confidence intervals) for algal growth were 18 (4.3 - 71.5) and 32 (13.9 - 50.9) mg/L, respectively, and for percent inhibition were 18 (5.3 - 62.6) and 20 (5.0 - 79.3) mg/L, respectively. The 72 and 120 hour NOECs for algal growth were both 15 mg/L.

Executive summary:

The phytotoxicity of the test material to a freshwater green alga, Selenastrum capricornutum Printz, was investigated over a five day period in a study conducted under GLP conditions and in accordance with the standardised guideline OECD 201 and the Toxic Substances Control Act Test Guidelines.

During the study, algae, at an initial cell density of 10 000 cells/mL, were exposed to nominal concentrations of the test material of 0 (medium control) 1.88, 3.75, 7.5, 15, 30 and 60 mg/L under static conditions. The test flasks were incubated in an environmental growth chamber under continuous illumination with constant agitation. Growth (total cells/mL) and inhibition (percent) were determined.

Under the conditions of this study, the 72 and 120 hour EC50 values (and their 95 % confidence intervals) for algal growth were 18 (4.3 - 71.5) and 32 (13.9 - 50.9) mg/L, respectively, and for percent inhibition were 18 (5.3 - 62.6) and 20 (5.0 - 79.3) mg/L, respectively. The 72 and 120 hour NOECs for algal growth were both 15 mg/L.