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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 - 9 October 2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: control, solvent control and nominal concentrations of 0.53, 1.5, 4.3, 12, 35 and 100 mg/L
- Sampling method: samples for analysis were taken at 0 hrs from the excess test solutions and at 72 hrs from a single test replicate solution of each exposure (containing algae). All 72 hr samples were analysed post centrifugation.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal 1000 g/L primary stock solution of the test substance was prepared by the addition of 5g (nominal) of the test substance to 5 ml of dimethylformamide. 200 µL of this concentrated stock solution was then added to 2000 mL of culture medium to produce a top concentration of 100 mg/L with 100 µl/L of DMF. This stock solution was stirred vigorously overnight at approx. 30°C to aid the solubility of the test substance. This was then filtered with 0.2 µm GFC filter paper after which it was observed to be a clear and colourless solution.
One liter of each test solution was prepared by the addition of aliquots of the top concentration solution to sterile culture medium to final volumes of 1000 mL. The resultant solutions were clear and colourless. The control consisted of culture medium only. In all cases the final solutions contained nutrients. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: unicellular green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): Brixham Envirnmental Laboratory culture from strain ATCC 22662
- Age of inoculum (at test initiation): 4 day old culture of the alga in the exponential growth phase was used as inoculum for the test.
- Method of cultivation: The culture was grown in the medium and under the environmental conditions, described for the test. Laboratory cultures maintained under axenic conditions.

- Acclimation period: not relevant
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
72 hours
24 mg CaCO3/L
Test temperature:
The hourly temperature measurements, recorded automatically, remained within 20 ± 2°C
t=0h: 7.5 - 7.6
t=72h: 7.5 - 7.6
Dissolved oxygen:
not reported
not relevant
not applicable
Nominal and measured concentrations:
Nominal: dilution water control, solvent control, 0.53, 1.5, 4.3, 12.3, 35.1, 100 mg/L
Measured: dilution water control, solvent control, 0.57, 1.5, 3.7, 12, 32, 89 mg/L
The mean measured concentrations ranged from 85 - 10% of the nominal values, therefore based on the analytical results the nominal test substance concentrations were used for the calculation and reporting of the results.
Details on test conditions:
- Test vessel: conical flasks
- Material, size, headspace, fill volume: 250 mL, all-glass, containing 100 mL of test solution closed with foam bungs
- Initial cell density: 0.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates):
* 6 replicates each of the control and solvent control;
* 3 replicates of each concentration;

- Standard medium used: AAP
- Detailed composition:
NaHCO3: 15 mg/L
NaNO3: 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4: 1.044 mg/L
FeCl3.6H2O: 0.160 mg/L
Na2EDTA.2H2O: 0.3 mg/L
H3BO3: 0.186 mg/L
MnCl2.4H2O: 0.416 mg/L
ZnCl2: 0.00327 mg/L
CoCl2.6H2O: 0.001428 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O: 0.000012 mg/L

- Source/preparation of dilution water: AAP; according to the OECD 201 Guideline, formulated using Milli-RO water
- Culture medium different from test medium: No. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements:
* Microscopic obervations were made on samples taken from the control and each test substance concentration
* pH was measured at the beginning and at the end of the test using one replicate vessel (containing algae) from each test concentration.
* Temperature was continuously measured
* Light intensity was measured once during the study, in each of the four representative positions
* After 24, 48 and 72 hrs samples were removed from each test and blank vessel. The appropriate blank particle count was substracted from that of the test culture to obtain the cell density.
* At the end of the test, microscopic observations were made on samples taken from the control and each test substance concentration

- Photoperiod: continuous illumination
- illumination: "cool-white" of approx. 6000 lux. 5643 lux (by cosine receptor) at beginning of the study (with a range of ± 10%)
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth inhibition
- Determination of cell concentrations: At the beginning, cells were determined by eletronic particle counting, using a Coulter counter and counting at a lower threshold equivalent spherical diameter of approx. 2.3 µm
- effect calculated parameters: specific growth rate and yield

Reference substance (positive control):
Key result
72 h
Dose descriptor:
Effect conc.:
12 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
739 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
95% C.L.: 300-3900
Details on results:
- 72-h LOEC (based on yield) = 35 mg/L
- 72-h LOEC (based on growth rate) = 35 mg/L

- 72-h NOEC (based on yield) = 12 mg/L
- 72-h EC50 (based on yield) = 46 mg/L (95% C.L.: 40-51 mg/L)

- Exponential growth in the control (for algal test): the increase in biomass for the parent inoculum culture was monitored over its 4 day growth period and was considered to be in an exponential growth phase. The overall factor increase in cell particle density was 303.
- Observation of abnormalities (for algal test): the microscopic observations made at the end of the test showed that compared to the control, all of the algal cells sampled from the solvent control, 0.53, 1.5 and 4.3 mg/L test concentrations appeared normal. In the 12, 35 and 100 mg/L test concentrations some cells within the subsample were observed to be normal however others were observed to be enlarged.

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 highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, onesided, smaller).

The cell particle densities were examined by one-way analysis of variance and Dunnett's procedure to identify significant differences from the control.
Yield: A Weibull model was fitted to the percent effect and log concentration data.
Growth rate: A Probit model was fitted tothe percent effect and log concentration data.
Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular algal species Pseudokirchneriella subcapitata was performed with the test substance according to the OECD guideline 201 (GLP conditions). The EC50 for growth rate inhibition (72-h ErC50) was 739 mg/L. The 72-h NOEC for growth rate inhibition was determined to be 12 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

72 hr EC50 cell number (Pseudokirchnerella subcapitata) 739 mg/l (nominal). 
72 hr NOEC growth rate (Pseudokirchnerella subcapitata) 12 mg/l (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:
739 mg/L
EC10 or NOEC for freshwater algae:
12 mg/L

Additional information

Toxicity to algae and cyanobacteria has been tested with Pseudokirchnerella subcapitata over a 72 hour period at 100 mg/l. The 72h-EC50 (cell number) was 739 mg/l (nominal) and the 72h NOEC (growth rate) was 12 mg/l. The measured concentrations were higher than 80% of the nominal concentrations..