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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nine different concentrations of the test substance between nominal 0.39 and 100.00 mg per L nutrient medium, spaced by a factor of 2.0, were tested against one concurrent negative control (nutrient medium only).
- Sampling method: At the start and at the end of the incubation period samples of the test media were drawn and the concentrations of the test substance were determined in aliquots of the blank containing test substance, but no algae, and of one replicate of each of the test and control cultures. The sample was analysed in duplicate on the same day by HPLC.
- Sample storage conditions before analysis: The samples were analysed on the day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared by dissolving 111.5 mg of the test substance in 1 L of nutrient medium by ultrasonication for 15 minutes. Aliquots of this stock solution were diluted with nutrient medium to obtain lower concentrations, nominally spaced by a factor of 2. The preparations were made freshly before the start of the exposure.
- Eluate: no.
- Differential loading: no.
- Controls: For the negative control group only nutrient medium was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none.
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable.
- One blank, containing the highest test substance concentration, but no algae, was incubated under the same culture conditions for analysis of the test substance concentration and comparison with the algae cultures.
- The test was performed without adjustment of the pH of the test substance cultures.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum).
- Strain: ATCC (American Type Culture Collection) 22662.
- Source (laboratory, culture collection): LGC Promochem GmbH, Germany.
- Age of inoculum (at test initiation): /
- Method of cultivation: Stock cultures, initially cultivated from deep frozen algae, were continuously maintained in 250 mL conical flasks. each containing 100 mL nutrient medium, at a temperature of 23 °C under permanent light with an intensity of at least 6000 lux. In about weekly intervals 1 mL of the stock culture was diluted 100-fold with nutrient medium for precultivation and incubation is continued.

ACCLIMATION
- Acclimation period: Precultures were prepared by incubating a new stock culture for four days.
- Culturing media and conditions (same as test or not): same.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
/
Post exposure observation period:
Observation during the 72 h exposure period.
Hardness:
Not reported.
Test temperature:
23 +- 2
pH:
The test substance did slightly alter the pH of the test medium of 7.22 at the start of incubation.
pH 6.85 to 7.53 at the start.
pH 8.95 to 10.21 at 72 h.
Dissolved oxygen:
Not reported.
Salinity:
Fresh water was used.
Nominal and measured concentrations:
With algae:
Nominal concentrations: 0 - 0.39 - 0.78 - 1.56 - 3.13 - 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
Actual concentrations at the start: 0 - 0.41 - 0.72 - 1.56 - 3.15 - 6.27 - 12.5 - 24.9 - 50.1 - 101 mg/L.
Actual concentrations after 72 h: 0 - 0.08 - 0.05 - 0.21 - 0.29 - 0.54 - 2.32 - 7.62 - 25.3 - 76.6 mg/L.

Blank without algae:
Nominal concentrations: 100 mg/L.
Actual concentrations at the start: 101 mg/L.
Actual concentrations after 72 h: 95.1 mg/L.

The actual test substance concentrations determined at the start of the incubation were between 91.5 and 103.7 % of the nominal concentrations in the test substance cultures (100.6 % in the blank without algae).
After 72 hours of incubation the three highest test substance concentrations were between 30.6 and 76.0 % of the actual starting concentrations. The six lowest test substance concentration were between 7.0 and 19.8 % of the actual starting concentrations.
The actual test substance concentration of the blank without algae was 94.5 % of the actual starting concentration.
This result gives an indication for an uptake of the test substance by the algae or for adherence of the test substance to them. Therefore the calculation of the EC-values, the NOEC's and the presentation of the results was based on the actual concentrations determined at the start of the test as well as on the geometric means of the actual test substance concentrations determined at the start and at the end of the incubation.
In the blank, the test substance concentrations has been satisfactorily maintained to within 80 - 100 % of the initial concentrations throughout the duration of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks.
- Initial cells density: 100 000 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Sterile deionised water and sterile concentrated nutrient medium 9 + 1 was used.
- Conductivity of the deionised water: <5 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Photoperiod: 24 h.
- Light intensity and quality: 6000 lux. Wavelength of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic cell counter.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: n.a.
- Range finding study: not reported.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 101 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 101 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 101 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 101 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): During the 72 hours incubation period the cell density in the control cultures increased by a factor of ca. 91, corresponding to about 6.5 generations.
- Observation of abnormalities (for algal test): non.
- Any stimulation of growth found in any treatment: In all concentrations tested, the influence on algal growth was low and ranged from 14.5 % inhibition to 7.1 % enhancement.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none.
- Effect concentrations exceeding solubility of substance in test medium: no.
Results with reference substance (positive control):
Not relevant.
Reported statistics and error estimates:
Not relevant.

The results indicate an uptake of the test substance by the algae or an adherence of the test substance to them. Therefore the calculation of the NOECs and of the EC50 values was based on the actual concentrations determined at the start of the test as well as on the geometric means of the actual test substance concentrations determined at the start and at the end of incubation.

Validity criteria fulfilled:
yes
Conclusions:
The NOEC >= 101 mg/L.
The EC50 > 101 mg/L.
Executive summary:

A Pseudokirchneriella subcapitata growth inhibition test according to the directive 92/69/EEC Part C.3 was performed to determine the possible effects of the test substance on the growth of a unicellular green algal species.

In all concentrations tested, the influence on algal growth was low and ranged from 14.5 % inhibition to 7.1 % enhancement.

The NOEC >= 101 mg/L. The EC50 > 101 mg/L.

Description of key information

Key study. Test method according to EU C.3. GLP study. The 72h-EC50 was > 101 mg/L and the 72h-NOAEC was >/= 101 mg/L.

Key value for chemical safety assessment

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

Additional information