Methyl 3-aminopyrazinecarboxylate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April 2013 - 11 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 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

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:

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

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2 ml
Storage: Samples were stored in a freezer until analysis.

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

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 2 ml were taken from all test solutions for possible analysis. If not already 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 batch of Amiloride Compound 6 tested was a tan powder with a purity of 100% based on area percentage and was completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with individually prepared loading rates of 1.0, 10 and 100 mg/l. The loading rates of 1.0 and 10 mg/l were completely dissolved after 44 minutes of magnetic stirring. For the highest loading rates a 30- minute period of magnetic stirring was applied and was followed by a 5-minute treatment with ultrasonic waves to accelerate the dissolving of the test substance in the test medium. The obtained mixture containing undissolved material was stirred magnetically for a period of 6 minutes after which all test substance was completely dissolved. The test solutions prepared at 1.0 and 10 mg/l were clear and colourless whereas the highest test concentration was clear and slightly brown.

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

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10E4 cells/ml.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation):3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

0.24 mmol (24 mg CaCO3/L)

Test temperature:

22.0 - 22.9°C

pH:

t=0h: 8.2-8.3
t=72h: 8.1-8.3

Dissolved oxygen:

not measured

Salinity:

Not measured

Nominal and measured concentrations:

Nominal: 1.0, 10 and 100 mg/l

Measured: The actual concentration measured in 100 mg/l was in agreement with nominal during the first 24 hours of exposure (93-95% of nominal). Thereafter, it decreased to 75 mg/l (i.e. 75% of nominal) at the end of the test. Therefore, the Time Weight Average concentration was calculated to be 87 mg/l.

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1705000 cells/ml

Replicates:
6 replicates of the control and limit concentration,
3 replicates of the lower test concentrations.
1 replicate of each concentration without algae.
1 or 2 replicates of each test concentration for sampling purposes

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 93 to 102 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and 1.0, 10 and 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

87 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 87 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

87 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 87 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Details on results:

- Exponential growth in the control :yes
- Observation of abnormalities: no abnormalities observed
- Unusual cell shape: no
- Colour differences: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 0.97 mg/l with a 95% confidence interval ranging from 0.62 to 1.5 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman).

Statistical analysis of the data on growth rate was not needed as the effects recorded were not significant (<10%).

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

Any other information on results incl. tables

none

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No reduction of growth rate or inhibition of yield was recorded at any of the concentrations of Amiloride Compound 6 tested.

The EC50 for growth rate reduction (72h-ERC50) and yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 87 mg/l.

The 72h-NOEC for both growth rate reduction and yield inhibition was 87 mg/l.