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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Analytical monitoring:

yes

Details on sampling:

Test item concentrations were analytically determined; samples were taken from each treatment before the start of the test and after 48 hours. Duplicate samples were taken for further analysis if necessary. All samples were stored frozen immediately after sampling. The concentrations were analyzed in one of the duplicate test medium samples of all test concentrations from the sampling times of 0 and 48 hours.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 liter to give a 200 mg/L stock solution. A series of dilutions was made from this stock solution to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. The test media was prepared just before introduction of the daphnids (i.e. start of the exposure).

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield, UK in 2002. Since that time, the clone has been bred at Envigo Research Limited in reconstituted water of the quality identical to the water quality used in definitive test (with respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the definitive test. At the start of the test, the organisms used were less than 24 hours old and were not first brood progeny.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCo3

Test temperature:

21 - 22 °C

pH:

7.8 - 8.0

Dissolved oxygen:

8.7 - 9.0

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 (t=0h): - Measured concentrations (t=48h):

Details on test conditions:

TEST WATER
ISO reconstituted test water according to guideline. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated. The appearance of the test media was visually inspected at the start of the test and after 24 and 48 hours. The control and all test concentrations were observed to be clear colorless solutions.

MATERIALS
The test was performed in 150 mL glass beakers filled with 100 mL of test medium. The test vessels were covered to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

STUDY DESIGN
For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnid was 20 mL. The daphnids were randomly distributed to the test vessels at the initiation of the test.

EXPERIMENTAL CONDITIONS
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 18-22 °C with a maximum deviation of ± 1 °C. A 16-hour light to 8-Hour dark cycle with a 20 minute transition period was used. Light intensity during the light period was between 469 and 499 Lux. The daphnids were not fed during the test.

EFFECT PARAMETERS MEASURED
The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids that were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours exposure no immobilized daphnia were observed in the control, 6.25, 12.5, 25 and 50 mg/L test groups, 45% immobilization was observed in the 100 mg/L test group.

Results with reference substance (positive control):

48-h EC50: 0.54 mg/L. Periodical control test September 2015. The sensitivity of the test organisms was within the internal historical range.

Reported statistics and error estimates:

The 24 and 48-Hour EC50 values were calculated by Probit analysis using Linear Maximum-Likelihood regression using the ToxRat Professional computer software package (TOXRAT). The NOEC was determined using Fisher’s Exact Test using the ToxRat Professional computer software package (TOXRAT).

Immobilization and Observations of Daphnia magna

Nominal test item concentration	No. of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
[mg/L]		No.	[%]	No.	[%]
Control	20	0	0	0	0
6.25	20	0	0	0 (2A)	0
12.5	20	0 (1A)	0	0 (1A)	0
25	20	0	0	0 (1B)	0
50	20	0	0	0	0
100	20	0	0	9 (4B)	45

Values in parenthesis: number of test animals with adverse effects:

A: Trapped at the suface

B: Reduced mobility

Validity criteria fulfilled:

yes

Conclusions:

The 24 and 48-Hour EC50 of the test item was calculated to be greater than 100 mg/L based on nominal concentrations.

Executive summary:

The acute toxicity of the substance to Daphnia magna was determined in a 48 -hour static test according to the OECD Guideline for Testing of Chemicals No. 202 (2004) and the Method C.2 of Commission Regulation (EC) No. 440/2008.

The nominal test item concentrations tested were 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 85% to 101% 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. The 24 and 48-Hour EC50 of the test item was calculated to be greater than 100 mg/L based on nominal concentrations. After 48 hours exposure no immobilized daphnia were observed in the control, 6.25, 12.5, 25 and 50 mg/L test groups groups, 45% immobilization was observed in the 100 mg/L test group. The 48 -hour EC50 of the test item was calculated to be greater than 100 mg/L based on nominal concentrations.

Description of key information

The 48-h EC50 value is 100 mg/L in aquatic invertebrates (D. magna).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

100 mg/L

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD guideline No 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours under static conditions. Analytical confirmation of nominal test concentrations showed that all test concentrations remained well within ±20% of nominal concentrations throughout the test. Therefore, effect concentrations are expressed as nominal. Immobilization was recorded after 24 and 48 hours exposure. After 48 hours exposure no immobilized daphnia were observed in the control, 6.25, 12.5, 25 and 50 mg/L test groups, 45% immobilization was observed in the 100 mg/L test group. Based on these findings the 48-h EC50 value was determined to be >100 mg/L.