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

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:

18 October 1993 to 15 July 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

No analysis of test solutions.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Toxic Substances Control Act Test Guidelines

Deviations:

not specified

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

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 ± 1 °C

pH:

5.3 to 7.7 (without algae); 5.3 to 9.1 (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
- 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 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.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 8.9 to 65.0 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30 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.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 15.4 to 58.0 mg/L

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 Concentration (mg/L)	Time (hours)
	24	48	72	96	120
60	7460 7700 8707	5880 6493 7067	8827 7000 9053	8340 4453 6813	2087 807 1613
30	13093 14647 13513	36427 36827 38973	174400 134667 168733	459333 468667 592267	1153867 1082400 1688133
15	13820 16753 17340	41573 51653 44093	168333 194200 136933	690133 750000 632800	2204667 2221467 2072800
7.5	15000 14220 15607	46733 46720 62907	114867 128600 268000	384533 367733 931467	1935200 1508933 2801733
3.75	14120 16967 21227	41187 55493 54107	168067 236000 186733	592533 837733 737200	2219067 2474400 2354000
1.88	18760 19120 18980	58640 57467 58027	190533 217200 188067	849067 958400 805600	2422000 2863200 2282133
Control	15280 17807 16673 15220 16947 18453	46560 47520 48880 46520 49760 55347	200867 176533 191400 154667 212267 224400	667733 669600 747600 598133 724000 770400	2134533 2120000 1912133 1761733 2053333 2007200

 

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

Nominal Concentration (mg/L)	Statistics	Time (hours)
		24	48	72	96	120
60	Mean SD CV	7956* 661.5 8.3	6480* 593.4 9.2	8293* 1125.8 13.6	6536* 1958.2 30.0	1502* 647.2 43.1
30	Mean SD CV	13751* 803.5 5.8	37409* 1369.5 3.7	159267 21491.8 13.5	506756 74201.7 14.6	1308133* 331024.0 25.3
15	Mean SD CV	15971 1885.9 11.8	45773 5245.8 11.5	166489 28677.9 17.2	690978 58604.6 8.5	2166311 81417.5 3.8
7.5	Mean SD CV	14942 695.1 4.7	52120 9341.5 17.9	170489 84725.8 49.7	561244 320731.9 57.1	2081956 658776.0 31.6
3.75	Mean SD CV	17438 3576.7 20.5	50262 7890.2 15.7	196933 35096.5 17.8	722489 123260.2 17.1	2349156 127735.6 5.4
1.88	Mean SD CV	18953 181.5 1.0	58044 586.9 1.0	198600 16155.2 8.1	871022 78730.5 9.0	2522444 303276.2 12.0
Control	Mean SD CV	16730 1308.6 7.8	49098 3319.9 6.8	193356 25145.4 13.0	696244 63333.1 9.1	1998156 141327.0 7.1

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 Inhibition of Growth in Comparison to the Control at Each 24 Hour Interval

Nominal Concentration (mg/L)	% Inhibition
	24 h	48 h	72 h	96 h	120 h
60	130.4	114.5	104.7	101.6	100.8
30	44.3	33.6	23.1	25.1	30.5
15	11.3	9.2	12.7	5.9	-2.5
7.5	26.6	1.1	7.4	15.6	6.0
3.75	-10.5	-4.9	-2.7	-3.2	-10.9
1.88	-33.0	-25.5	-10.0	-18.1	-23.7
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 hour EC50 value and 95 % confidence interval for algal growth was 37 (8.9 to 65.0) mg/L. The 72 hour NOEC was 30 mg/L. Results are based on nominal concentrations.

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 hour EC50 value and 95 % confidence interval for algal growth was 37 (8.9 to 65.0) mg/L. The 72 hour NOEC was 30 mg/L. Results are based on nominal concentrations.