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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
(Range-finding study) 5 January to 8 February 1999, (Definitive study) 24 May to 27 May 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(Annex V of Council Directive 67/548/EEC)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Date of inspection: 23 March 1998 Date of Signature: 21 July 1998)
Analytical monitoring:
yes
Details on sampling:
- Concentrations, sampling method: Water samples were taken from the control and the saturated solution (prepared from an initial test material dispersion at a concentration of 100 mg/I (replicates R1 - R3 and R4 - R6 pooled)) at 0 and 72 hours for quantitative analysis.

- Sample storage conditions before analysis: Duplicate samples were taken at each occasion and stored frozen (approximately -20°C) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

Due to the low aqueous solubility and high purity of the test material, the test concentration used in the definitive study was a saturated solution prepared from an initial dispersion at a concentration of 100 mg/l.
An amount of test material (200 mg) was added to the vortex created in 2 litres of culture medium to give an initial test material dispersion at a concentration of 100 mg/l. The mixture was stirred for a period of 47 hours with a vortex depth of approximately 25% of the water column height. After 47 hours the stirring was stopped and the mixture allowed to stand for 1 hour prior to the removal of the saturated solution by filtration through 0.2 µm filters.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green Algae

- Strain: CCAP 276/20

- Source (laboratory, culture collection):
Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Centre of Algae and Protozoa (CCAP), Institute of Freshwater Ecology,Ferry House, Ambleside, Cumbria, United Kingdom.

- Method of cultivation:
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and continuous illumination at 21 ± 1ºC.

- Culturing media and conditions (same as test or not):
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable.

Hardness:
Not reported.
Test temperature:
24 ± 1ºC.

pH:
7.3 (0 h), 8.6 - 10.5 (72 h)
Dissolved oxygen:
Not reported.
Salinity:
Not reported.
Nominal and measured concentrations:
(Range-finding test) 10, 100 mg/l

(Definitive test) 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed.

- Material, size, headspace, fill volume: 250 ml glass conical flasks.

- Aeration: No.

- No. of organisms per vessel: 2.16 x 1E06 cells per ml.

- No. of vessels per concentration (replicates): 6.

- No. of vessels per control (replicates): 3.

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
Stock Solutions
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l


OTHER TEST CONDITIONS
- Adjustment of pH: The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Photoperiod: Continuous illumination for 72 hours.

- Light intensity and quality: intensity approximately 7000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisiser II Particle Counter.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 and 100 mg/l.

- Results used to determine the conditions for the definitive study: The results showed no effect on growth in the saturated solutions prepared from
initial test material dispersions at concentrations of 10 and 100 mg/l.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.71 mg/L
Nominal / measured:
estimated
Conc. based on:
dissolved
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.71 mg/L
Nominal / measured:
estimated
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.71 mg/L
Nominal / measured:
estimated
Conc. based on:
dissolved
Basis for effect:
other: biomass, growth rate
Details on results:
Neither the growth or the biomass of Scenedesmus subspicatus (CCAP 276/20) was affected by the presence of the saturated solution prepared from an initial test material dispersion at a concentration of 100 mg/I over the 72-hour exposure period.

- Exponential growth in the control (for algal test): yes

- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures.

- Adherence to test vessels: At the lower concentration of 1.0 mg/I the unshaken/unsonicated vessel stability samples were considered to show possible evidence of insolubility or adherence to glass. However, the results obtained cannot directly indicate the stability of the prepared saturated solution as these were obtained from samples produced using a different method of preparation to that employed in the definitive study.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:

- Verification of test concentrations: Chemical analysis of the saturated solution (prepared from an initial test material dispersion at a concentration of 100 mg/l) at 0 and 72 hours showed measured concentrations to be below the limit of quantitation of the analytical method. 

Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Not reported.


Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and based on an estimate of the dissolved concentration of the test material (limit of quantitation) gave a 72-Hour EC50 value of greater than 0.71 mg/l. Correspondingly the No Observed Effect Concentration was 0.71 mg/l.
Executive summary:

Methods

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus.The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test"

referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

 

Procedure

Following a preliminary range-finding study,Scenedesmus subspicatuswas exposed to a saturated solution prepared from an initial test material dispersion at a concentration of 100 mg/I (six replicate flasks) for 72 hours, under constant illumination and shaking at a

temperature of 24 ± 1ºC.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter® Multisizer II Particle Counter.

Results

Analysis of the saturated solution (prepared from an initial test material dispersion at a concentration of 100 mg/I) at 0 and 72 hours showed the measured test concentrations to be below the limit of quantitation of the analytical method employed. This does not infer that no test material was in solution but that the dissolved concentration of test material (ie bioavailable to the algae) was below the limit of quantitation which was assessed down to 0.71 mg/l. It was considered unnecessary and unrealistic to test saturated solutions prepared from initial test material dispersions at concentrations in excess of 100 mg/l.

Therefore the 72-Hour EC50 based on the limit of quantitation was greater than 0.71 mg/I and correspondingly the No Observed Effect Concentration was 0.71 mg/l.

Description of key information

A study was performed to assess toxicity on growth of aquatic algae for TXAX. Under the test condition, the dissolved concentration (ie. bioavailable to the test organisms) of the test material was below the limit of quantitation which was assessed down to 0.71 mg/L. It was considered unnecessary and unrealistic to test saturated solutions prepared from initial test material dispersions at concentrations in excess of 100 mg/L. Therefore the 72-Hour EC50 based on the limit of quantitation (growth rate) was greater than 0.71 mg/L.

Key value for chemical safety assessment

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

Additional information

Only one study is available for this endpoint and this will be submitted as a key study for the substance. The 72-Hour EC50 based on the limit of quantitation was greater than 0.71 mg/L.

A study was performed to assess toxicity of the test material to aquatic algae. The method followed that described in the OECD TG 201 and conducted at a GLP-facility with a reliability of the study 1, according to the scoring system of Klimisch et al., 1997.