Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

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:

02 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

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:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

EC Number:

215-947-7

EC Name:

Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Cas Number:

1458-18-0

Molecular formula:

C6H5Cl2N3O2

IUPAC Name:

methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Identification: Amiloride Compound 7
Molecular formula: C6H5Cl2N3O2
Molecular weight: 222.03
CAS Number:1458-18-0
Description: Tan powder
Batch: 206-2
Purity/Composition: 99.7 area%
Test substance storage: In refrigerator (2-8°C) protected from light, desiccated
Stability under storage conditions: Stable
Expiry date: 11 May 2014 (Retest date)

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. In addition, the filter containing undissolved residue was retained for possible analysis.

Frequency: at t=0 h and t=48 h
Volume: 2.4 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 2.4 ml were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface). No correction was made for the purity/composition of the test substance.

The batch of Amiloride Compound 7 tested was a tan powder with a purity of 99.7 area% and not completely soluble in test medium at the loading rates initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l applying a two-day period of magnetic stirring to ensure maximum dissolution in the test medium. The resulting dispersion was subsequently filtered through a 0.45 µm membrane filter (Whatman, RC55) to remove the undissolved fraction of test substance. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

Note that due to light sensitivity of the test substance all handlings were performed under dimmed light and the 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 < 24 hours, from parental of daphnids 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:

19.7-20.3

pH:

7.8-8.1

Dissolved oxygen:

8.8-9.3

Salinity:

not measured

Nominal and measured concentrations:

nominal concentrations: 10, 18, 32, 56 and 100% of a filtrate prepared at a loading rate of 100 mg/l.

The actual concentrations, measured in the solutions containing 10, 18, 32 and 100% of the filtrate, were 0.70, 1.2, 2.2 and 7.0 mg/l, respectively, and remained stable during the exposure period (81-82% of initial). Therefore, the effect parameters were reported in terms of initially measured 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.:

1.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.2-1.5 mg/l

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.1-1.3

Details on results:

-Behavioural abnormalities: no
-Other biological observations: none
-Mortality of control: 5%
-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:

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 initially measured test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Amiloride Compound 7 did not induce acute immobilisation of Daphnia magna at an initial measured concentration of 0.70 mg/l after 48 hours of exposure. The EC 50 at 48 hours was 1.2 mg/L (95% CL: 1.1-1.3 mg/L).

The 48h-EC50 was 1.2 mg/l based on initial measured concentrations (95% confidence interval between 1.1 and 1.3 mg/l).