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

Study period:

08 January 2016 to 09 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

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

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: 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.

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

24 ± 1 ºC

pH:

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.5 at 96 hours. The pH values in the treated culture ranged from 7.2 at 0 hours to to 8.8 at 96 hours.

Nominal and measured concentrations:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 92% to 98% of nominal indicating that the test system had been correctly dosed. A concentration dependent decline in measured concentration occurred at 96 hours in the range of 51% of nominal at 6.25 mg/L, through to 97% of nominal at 100 mg/L. Given this decline it was considered appropriate to calculate the results of the study based on the geometric mean measured test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks plugged with polyurethane foam stoppers.
- Aeration: Orbital shaker (100 - 150 rpm)
- Renewal rate of test solution (frequency/flow rate): none (static test)
- Initial cells density: 5000 cells/mL
- Control end cells density: 8.2 x 10E5 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Reconstituted test water (ASTM medium) prepared according to OECD Test Guideline No 201 was used for algal cultivation and testing)

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: The pH of the culture medium was adjusted to 7.5 at preparation.
- Photoperiod: cultures were continuously illuminated
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7184 Lux (range: 7110 to 7320 Lux). The light intensity over the incubation area was within a ±15% deviation from the average light intensity.
- Determination of cell concentrations: electronic particle counter (Coulter Multisizer particle Counter)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test): 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.
- Unusual cell shape:
- Colour differences: After the 96-Hour test period the 4.3, 9.5, 21 and 46 mg/L test cultures were observed to be bright green dispersions whilst the 98 mg/L test cultures were observed to be green dispersions. Control replicate R1 was observed to be an extremely pale green dispersion whilst replicates R2-R6 were observed to be bright green dispersions.
- Flocculation:
- Adherence to test vessels:
- Aggregation of algal cells:
- Other:
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colourless solutions.
- Effect concentrations exceeding solubility of substance in test medium:

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the last positive control test prior to this study performed between 7 December 2015 and 10 December 2015, gave an ErC50 value of 1.5 mg/L. This value was within the historical range of 0.82 – 1.5 mg/L obtained between 2010 and 2015.

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).

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Time Point (hours)	Response Variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
72	Growth Rate	54	91	>98	21
	Yield	23	34	67	21
	Biomass	24	37	78	21
96	Growth Rate	98	>98	>98	21
	Yield	23	40	>98	21
	Biomass	23	37	86	21

It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.

In terms of growth rate at 72 and 96 hours, there were no statistically significant differences (P≥0.05), between the control, 4.3, 9.5 and 21 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 21 mg/L. Similarly, the NOECs based on yield and biomass integral (AUC) was 21 mg/L.

 The following data show that the cell concentration of the control cultures increased by a factor of 164 after 72 hours:

- Mean cell density of control at 0 hours : 5.00 x 10e3 cells per mL

- Mean cell density of control at 72 hours : 8.20 x 10e5 cells per mL

This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

The mean coefficient of variation for section by section specific growth rate for the control cultures after 72 hours was 11% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:

yes

Conclusions:

A study was performed to assess the effect of test item 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 Guideline 201 and EU Method C.3. The test substance 72 and 96 hour ErC50 values were >98 mg/L. The NOEC was 21 mg/L at both 72 and 96 hours.

Executive summary:

A study was performed to assess the effect of test item 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 Guideline 201 and EU Method C.3.

Following a preliminary range-finding test and initial experiments, 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.

The test substance 72-hour EC50 values were >98 mg/L (growth rate), 67 mg/L (yield) and 78 mg/L (biomass). The 96-hour EC50 values were >98 mg/L (growth rate), >98 mg/L (yield) and 86 mg/L (biomass). The NOEC for all response variables was 21 mg/L.

Description of key information

A study was performed to assess the effect of test item 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 Guideline 201 and EU Method C.3. The test substance 72 and 96 hour ErC50 values were  >98 mg/L. The NOEC was 21 mg/L at both 72 and 96 hours.  

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

21 mg/L

Additional information

The measured concentration at the highest test concentration was 98% of the nominal concentration at the start of the exposure and 97% at the end of the exposure (96h). In this case, the effect level can be based on the nominal test concentration. The ErC50 value could not be determined in this study and is above the highest test concentration. Therefore, the ErC50 value must be greater than 100 mg/L. This effect level is taken into consideration in the derivation of the classification for environmental hazard in accordance with Regulation (EC) No. 1272/2008.