3-aminopyrazine-2-carboxylic acid

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:

18 March 2013 to 20 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-aminopyrazine-2-carboxylic acid

EC Number:

226-558-7

EC Name:

3-aminopyrazine-2-carboxylic acid

Cas Number:

5424-01-1

Molecular formula:

C5H5N3O2

IUPAC Name:

3-aminopyrazine-2-carboxylic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Molecular formula: C5H5N3O2
Molecular weight: 139.11
CAS Number: 424-01-1
Description: Tan powder
Batch: 203-2
Purity/Composition: 100 area%
Test substance storage: In refrigerator (2-8°C) protected from light, desiccated
Stability under storage conditions: Stable
Expiry date: 13 March 2014 (Retest date)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Hygroscopic: Yes, store desiccated
Volatile: No
Reactivity: Reactive to light, moisture and oxygen
Test substance handling: No specific handling required for moisture and oxygen. Use amber-coloured glassware or wrap container in aluminium-foil
Stability in water: Unknown
Solubility in water: 2.4 g/l

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 5 tested was a tan powder with a purity of 100 area% and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with a concentration of 100 mg/l applying 20 minutes of magnetic stirring followed by a 15-minute treatment with ultrasonic waves to accelerate the dissolving of the test substance in the test medium. The pH was adjusted to 6.5 using 1M NaOH (Merck, Darmstadt, Germany). The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were clear and colourless to clear and slightly yellow in the highest test concentration.

Note that the preparation was performed under dimmed light.

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 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:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

-

Post exposure observation period:

no

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

19.2-20.6

pH:

6.8-8.0

Dissolved oxygen:

8.8-9.3

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations: 0.1, 1.0, 10, and 100 mg/l.

The actual concentrations were at the level of nominal (110% of nominal) and remained stable during the test (99% of initial). Consequently, the effects parameters were reported in terms of analytical confirmed nominal concentrations.

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 100 ml, all-glass
Medium: Adjusted ISO medium
Number of daphnids : 20 each for the control and highest (limit) test concentration, 10 each for the remaining concentrations (combined range-finding test).
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 daphnids : Within 20 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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Details on results:

- Behavioural abnormalities:
- Other biological observations: none
- Mortality of control: no
- Other adverse effects control: no

Results with reference substance (positive control):

- Results with reference substance valid? yes

- EC50: 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:

No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum concentration tested).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Amiloride Compound 5 did not induce acute immobilisation of Daphnia magna at an analytical confirmed nominal concentration of 100 mg/l after 48 hours of exposure.

The 48h-EC50 was beyond the range tested, i.e. exceeded an analytical confirmed nominal concentration of 100 mg/l.