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

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:

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

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:

- Name of test material (as cited in study report): Amiloride Compound 7
- Description: Tan powder
- Storage condition of test material: In refrigerator protected from light, desiccated
- Other: Sample is hygroscopic and reactive to light and oxygen

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Purity/composition correction factor required: No
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: Insoluble

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

Frequency:at t=0 h, t=24 h and t=72 h
Volume: 2.0 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 2.0 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 7 tested was a tan powder with a purity of 99.7 area% and not completely soluble in test medium at the loading rate 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 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 or yellow light and the glassware 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:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

0.24 mmol(24 mg CaCO3/L)

Test temperature:

between 21.7 and 22.9°C

pH:

T=0 h between 8.1-8.2
T=72 h between 7.9-8.1

Nominal and measured concentrations:

Nominal concentrations: control and 0.32, 1.0, 3.2, 10 and 32% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg/l
Measured concentrations:The actual test concentrations at the start of the test were 0.028, 0.072, 0.22, 0.64 and 2.1 mg/l in the solutions containing 0.32, 1.0, 3.2, 10 and 32% of the filtrate, respectively. The actual concentrations in solutions containing 3.2 to 32% of the filtrate decreased to 19-54% of initial at the end of the test. The measured concentration in the remaining test groups were below the limit of detection of the analytical method (LOD=0.0044 mg/l) at the end of the test. Calculated Time Weight Average concentrations were 0.028, 0.072, 0.22, 0.64 and 2.1 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: 2336000 cells/ml
Replicates:
6 replicates of the control,
3 replicates of each test concentration,
2 replicates of the undiluted filtrate without algae,
1 replicate 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 91 to 99 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC10, 72 h EyC10, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: control and 0.32, 1.0, 3.2, 10 and 32% of a 0.45 μm filtered solution prepared at a loading rate of 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.:

0.028 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:

EC10

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.015 -0.15 mg/l TWA

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.10 - 0.91 mg/l TWA

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: -

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 0.019- 0.034 mg/l TWA

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI:0.068 - 0.12 mg/l TWA

Details on results:

Exponential growth in the control (for algal test): yes
Obeservation of abnormalities (for algal test): no abnormalities observed.
Unusual cell shape: no
Colour differences: no

Results with reference substance (positive control):

Validity criteria met: yes
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). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test substance.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Amiloride Compound 7 reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 0.12 mg/l and higher.
The EC50 for growth rate reduction (72h-ERC50), based on TWA concentrations, was 0.30 mg/l with a 95% confidence interval ranging from 0.10 to 0.91 mg/l.
The EC50 for yield inhibition (72h-EYC50), based on TWA concentrations, was 0.092 mg/l with a 95% confidence interval ranging from 0.068 to 0.12 mg/l.
The 72h-NOEC for both growth rate reduction and yield inhibition was, based on the TWA concentration, 0.028 mg/l.