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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
Algae were not microscopically examined for any malformations at the end of the study. Evaluation: No effects on growth of algae were observed up to a highest concentration of 100 mg/l tested and it was therefore expected that algae were not malformed. He
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: control and 100 mg/l
- Sampling method: 10 ml from the approximate centre of the test vessels at t=0h, t=24h and t=72h
- Sample storage conditions before analysis: stored in a freezer
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a concentration of 100 mg/l. No special treatment other than thorough mixing was necessary to completly dissolve the test substance in test medium. The lower test concentrations were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless.
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 (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continously aerated and exposed to light in a climate room at a temperature of 21-24°C

- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): The stock culture medium is M1 (according to the NPR 6505, formulated using Mili-Ro water); the pre-culture and culture medium is M2 (according to the OECD 201 guideline, formulated using Mili-Q water
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
21 - 24 °C
7.9 - 8.3
Nominal and measured concentrations:
See details on analytical meetings
Details on test conditions:
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Initial cells density: 10000 cells/ml
- Control end cells density: 1940100 cells/ml
- No. of vessels per concentration (replicates): 6 replicates of the highest test concentration, 3 replicates of the lower concentrations, 1 replicate of each test concentration without algae, 1 replicate of of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control and 1 replicate of the control without algae

- Standard medium used: yes

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continous
- Light intensity and quality: 83 to 88 ¿E.m-2.s-1 using TLD-lamps of the type ¿Cool-white¿ of
30 Watt

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 0, 24, 48 and 72 h
- Determination of cell concentrations: spectrophotometer

- Spacing factor for test concentrations: 10 (0.1, 1, 10 and 100 mg/l)
- Range finding study: the study is a combined limit/rang finding study
Reference substance (positive control):
potassium dichromate
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
loading rate
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Algae were not microscopically examined for any malformations at the end of the study, see guideline deviation.
- Any stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: for yield inhibition (EyC50: 0-72h) was 0.65 mg/l with a 95% confidence interval ranging from 0.44 to 0.97 mg/l.
Validity criteria fulfilled:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of CN-nitcal tested.
The EC50 for both growth rate reduction and yield inhibition based on nominal concentrations exceeded 100 mg/l, the regulatory limit concentration.
The NOEC for growth rate reduction and yield inhibition was 100 mg/l.

Description of key information

A reliable study with nitric acid, ammonium calcium salt showed that the substance was not inhibitory at the highest concentration tested, thus 72 hr EC50 was >100 mg/L. The NOEC was determined to be 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

A reliable study with the algae species, Pseudokirchneriella subcapitata, according to OECD 202 guidelines was performed. The 72 -hr EC 50 based on exposure to nitric acid, ammoium calcium salt was determined to be >100 mg/L. No reduction of growth rate or inhibition of yield was recorded at any of the concentrations of the substance tested. The NOEC for growth rate reduction and yield inhibition was 100 mg/L. Samples were taken for analytical measurement, but it was decided not to include analytical confirmation of actual exposure concentrations. Possible analyses could be based on Calcium, Ammonium and/or Nitrate. However, analysis of actual exposure concentrations was not performed as the test substance consisted of inorganic salts, which are partly present in the test medium (Calcium and Ammonium) or could only be analysed by a non-specific spectrophotometric analytical method (Nitrate). Results were based on nominal concentrations and as the test substance was well soluble and the inorganic salts present in the test substance are all known to be stable in aquatic media.