Methyl 3-aminopyrazinecarboxylate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April 2013 - 25 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-aminopyrazinecarboxylate

EC Number:

240-387-5

EC Name:

Methyl 3-aminopyrazinecarboxylate

Cas Number:

16298-03-6

Molecular formula:

C6H7N3O2

IUPAC Name:

methyl 3-aminopyrazine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Amiloride Compound 6
- Description: Tan powder
- Storage condition of test material: In refrigerator (2-8°C) protected from light, desiccated
- Other: Reactive to light, moisture and oxygen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0 h and t=48 h
Volume: 2 ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, singular reserve samples of 2 ml were taken for possible analysis. If not used, these samples were stored in a freezer.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Amiloride Compound 6 tested was a tan powder with a purity of 100% based on area percentage and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with individual loading rates ranging from 1.0 to 100 mg/l. A 30 and a 90-minute period of magnetic stirring were applied to accelerate the dissolving of the test substance in the medium, in the combined limit/range-finding test and the final, respectively. The final test solutions besides the highest test concentration were clear and colourless. The highest test concentration was clear and slightly yellow.

Note that due to the light sensitivity of the test substance all handlings were performed under dimmed light and glassware was either amber coloured or covered in aluminium foil.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of the batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

18.8 - 21.0 °C

pH:

7.8-8.1

Dissolved oxygen:

8.9-9.4 mg/l

Salinity:

not measured

Nominal and measured concentrations:

nominal concentrations: 10, 18, 32, 56 and 100 mg/l.

The actual concentrations, measured in the solutions containing 32, 56 and 100 mg/l, were at the level of nominal (100-104% of nominal) and remained stable during the test (91-93% of initial). Therefore, the effects parameters were reported in terms of analytically confirmed nominal concentrations

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessel: 100 ml, all glass
Medium: Adjusted ISO-medium
Number of daphnids: 20 per group
Loading: 5 per vessel containing 80 ml of test solution
Light: the test was performed under dimmed light conditions
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of the daphnids: within 18 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

analytically confirmed nominal

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 65-87 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

analytically confirmed nominal

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 61-78 mg/L

Details on results:

- Behavioural abnormalities: no
- Other biological observations: none
- Immobility of control: no immobility was observed.
- Other adverse effects control: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- The 48h-EC50 was 0.53 mg/l with a 95% confidence interval between 0.49 and 0.62 mg/l.

Reported statistics and error estimates:

The EC50-value was calculated at 24 and 48 hours of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Any other information on results incl. tables

none

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Amiloride Compound 6 did not induce acute immobilisation of Daphnia magna at an analytical confirmed nominal concentration of 32 mg/l after 48 hours of exposure. At 48 hours the EC50 was reported as 68 mg/l (95% CL: 61-78 mg/L)(nominal)

The 48h-EC50 was 68 mg/l based on analytically confirmed nominal concentrations (95% confidence interval between 61 and 78 mg/l).