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Ecotoxicological information

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:
18 March 2013 to 21 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
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, 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, singular 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 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 brown in the highest test concentration.

Note that the preparations were performed under dimmed light due to the light sensitivity of the test substance.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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
Remarks on exposure duration:
-
Post exposure observation period:
none

Test conditions

Hardness:
0.24 mmol(24 mg CaCO3/L)
Test temperature:
21.1-22.9
pH:
7.0-7.7
Dissolved oxygen:
not measured
Salinity:
not measured
Nominal and measured concentrations:
Nominal: 0.10, 1.0, 10 and 100 mg/l

Measured: The actual concentrations were at the level of nominal (109% of nominal) and remained stable during the test (101% of initial). Consequently, the effects parameters were reported in terms of analytically confirmed nominal concentrations.
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: 2045000 cells/ml
Replicates:
6 replicates of the control and the highest concentration
3 replicates of each lower test concentration
1 replicate of each concentration without algae

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 94 to 101 µE.m-2.s-1

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and 0.1, 1.0, 10 and 100 mg/l
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: -
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: -
Duration:
72
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: -
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no

- Any stimulation of growth found in any treatment: yes, very slight in 10 mg/l
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 the effect was >10% and 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).

No EC50-values 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:
No reduction of growth rate was recorded at any of the concentrations of Amiloride Compound 5 tested (72h-NOERC is 100 mg/l). Yield was significantly inhibited at an analytical confirmed nominal concentration of 100 mg/l.

The EC50 for both growth rate reduction (72h-ERC50) and yield inhibition (72h-EYC50) were beyond the range tested, i.e. exceeded an analytical confirmed nominal concentration of 100 mg/l.