2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD UK

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 96 hours for quantitative analysis. All samples were stored frozen immediately after sampling. Duplicate samples were taken at 0 and 96 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 liter to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. The test media were prepared just before inoculation with algal suspension. An aliquot (500 mL) of each of the test concentrations was separately inoculated with 3.9 mL of algal suspension to give a nominal cell density of approximately 5E+03 cells/mL.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

15 mg/L as CaCO3

Test temperature:

24 ± 1 °c

pH:

7.8 - 7.9

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L (based on range-finding test)
- Measured concentrations: <0.98, 5.80, 11.7, 23.4, 46.0, and 89.5 mg/L

Details on test conditions:

PREPARATION OF ALGAL TEST CULTURES
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 to 1E+05 cells/mL. The test was started using a nominal algal cell density of 5000 cells/mL. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Multisizer particle Counter).

TEST WATER
prepared according to guidelines. The culture medium used for tests was the same as that used to maintain the stock culture.

MATERIALS AND TEST DESIGN
250 mL Erlenmeyer flasks were used per replicate containing 100 mL of test solution. Each test flask was plugged with a polyurethane foam bung. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. The test design included three replicates per test concentration and six replicates of the control.

EXPERIMENTAL CONDITIONS
The test flasks were incubated (INFORS Multitron® Version 2 incubator) at a temperature of 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) by warm white lighting (380-730 nm). The mean measured light intensity at the level of the test solutions was approximately 7066 Lux (range: 6850 to 7180 Lux). The light intensity over the incubation area was within a ± 15 % deviation from the average light intensity.

DETERMINATION OF ALGAL BIOMASS
Samples were taken at 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5E+03 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded. The inhibition of algal growth, the percentage inhibition of biomass, the increase in biomass over the exposure period (yield) and the average specific growth rate for a specified period were determined.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: could not be calculated

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(yield)

Remarks on result:

other: 95% C.L.: 29-36 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(yield)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(yield)

Details on results:

Observations were made on the test cultures throughout the duration of the test. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 96-Hour test period all control, 6.25 and 12.5 mg/L test cultures were observed to be bright green dispersions. The 25 mg/L test cultures were green dispersions, the 50 mg/L test cultures were extremely pale green dispersions whilst the 100 mg/L test cultures were clear colorless solutions. All test and control cultures were inspected microscopically at 96 hours. After 96 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5 and 25 mg/L. Lots of cell debris was observed in the 50 mg/L test cultures whilst no intact cells were observed in the 100 mg/L test cultures.

In terms of growth rate at 72 hours, there were no statistically significant differences (P≥0.05), between the control, 6.25 and 12.5 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 12.5 mg/L. At 96 hours there were no statistically significant differences (P≥0.05), between the control, 6.25, 12.5 and 25 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 25 mg/L.

In terms of yield at 72 hours, there were no statistically significant differences (P≥0.05), between the control, 6.25 and12.5 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 12.5 mg/L. At 96 hours there were no statistically significant differences (P≥0.05), between the control, 6.25, 12.5 and 25 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 25 mg/L.

In terms of biomass integral (AUC) at 72 and 96 hours, there were no statistically significant differences (P≥0.05), between the control, 6.25 and 12.5 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral (AUC) was 12.5 mg/L.

Results with reference substance (positive control):

ErC50: 1.5 mg/L. Periodical control test performed between 7-10 December 2015. The sensitivity of the test organisms was within the internal historical range.

Reported statistics and error estimates:

Statistical analysis of the growth rate, yield and biomass integral data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

Inhibition of growth rate, yield and biomass integral (0 -72 hours)

Nominal test concentration (mg/L)	Growth rate		Yield		Biomass Integral (AUC)
	0-72h	Inhibition (%)	0-72h	Inhibition (%)	0-72h	Inhibition (%)
0 (control)	0.068	-	6.74E+05	-	1.19E+07	-
6.25	0.067	1	6.28E+05	7	1.13E+07	5
12.5	0.066	2	5.86E+05	13	1.11E+07	6
25	0.063	7	4.82E+05	28	9.34E+06	21
50	0.045	34	1.26E+05	81	6.60E+06	70
100	0.025	64	2.43E+04	96	1.20E+06	90

Inhibition of growth rate, yield and biomass integral (0 -96 hours)

Nominal test concentration (mg/L)	Growth rate		Yield		Biomass Integral
	0-96h	Inhibition (%)	0-96h	Inhibition (%)	0-96h	Inhibition (%)
0 (control)	0.060	-	1.61E+06	-	3.93E+07	-
6.25	0.060	1	1.54E+06	4	3.74E+07	5
12.5	0.059	1	1.50E+06	7	3.62E+07	8
25	0.058	4	1.29E+06	20	3.06E+07	22
50	0.040	34	2.28E+05	86	7.85E+06	80
100	0.021	64	3.44E+04	98	1.91E+06	95

Overview of reported effect concentrations

Time point (hours)	Response variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	95% Confidence Limits (mg/L)	NOEC (mg/L)
72	Growth rate	25	37	74	*	12.5
	Yield	15	20	32	29 – 36	12.5
	Biomass	17	23	38	34 – 43	12.5
96	Growth rate	27	39	73	*	25
	Yield	21	25	34	30 – 37	25
	Biomass	19	23	35	32 - 38	12.5

*It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h NOErC and 72-h ErC50 values are 12.5 mg/L and 74 mg/L, respectively in freshwater algae (P. subcapitata)

Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata in a 96-Hour static test. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201 (adopted 2006, corrected 2011) "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. Analysis of the test preparations at 0 and 96 hours showed measured test concentrations to range from 90% to 98% of nominal indicating that the test system had been correctly dosed and that the test item was stable under test conditions. Therefore the biological results of the study are based on the nominal concentrations only. Observations were made on the test cultures throughout the duration of the test. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 96-Hour test period all control, 6.25 and 12.5 mg/L test cultures were observed to be bright green dispersions. The 25 mg/L test cultures were green dispersions, the 50 mg/L test cultures were extremely pale green dispersions whilst the 100 mg/L test cultures were clear colorless solutions. All test and control cultures were inspected microscopically at 96 hours. After 96 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5 and 25 mg/L. Lots of cell debris was observed in the 50 mg/L test cultures whilst no intact cells were observed in the 100 mg/L test cultures. In terms of growth rate at 72 hours, there were no statistically significant differences (P≥0.05), between the control, 6.25 and 12.5 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 12.5 mg/L. At 96 hours there were no statistically significant differences (P≥0.05), between the control, 6.25, 12.5 and 25 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 25 mg/L In terms of yield at 72 hours, there were no statistically significant differences (P≥0.05), between the control, 6.25 and12.5 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 12.5 mg/L. At 96 hours there were no statistically significant differences (P≥0.05), between the control, 6.25, 12.5 and 25 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 25 mg/L In terms of biomass integral (AUC) at 72 and 96 hours, there were no statistically significant differences (P≥0.05), between the control, 6.25 and 12.5 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral (AUC) was 12.5 mg/L.

Description of key information

The 72-h ErC50 and ErC10 values are 74 mg/L and 25 mg/L, respectively in freshwater green algae (P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

74 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD guideline No 201 and in compliance with GLP criteria.

In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) at an initial cell concentration of ca. 5E+03 per mL, were exposed to nominal test substance concentrations of 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L for 96 hours under static conditions. The test was performed in 3 replicates per test concentration. Test concentrations were analytically verified and remained well within ± 20% of nominal throughout the test. After 24, 48, 72, and 96 hours, the algae were scored for the number of cells and for microscopic abnormalities.

After 72 hours exposure a significant inhibition of growth rate was observed at test concentrations of 25 mg/L (7%), 50 mg/L (34%) and 100 mg/L (64%). Inhibition of yield was also significant at 25 mg/L (28%), 50 mg/L (81%) and 100 mg/L (96%). Based on these findings the 72-h ErC50 and 72-h EyC50 values were determined at 74 mg/L and 32 mg/L, respectively. The 72-h ErC10 and 72-h EyC10 values were 25 mg/L and 15 mg/L, respectively. The NOEC for both endpoints was 12.5 mg/L.