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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Samples from control and test media were taken from freshly prepared uninoculated media at test start. At test end (72 hours) samples were taken from uninoculated replicate flasks incubated under the test conditions.
Samples were analysed without prior storage.
Details on test solutions:
REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
Aliquots of the test substance were dispersed in dilution medium. The bulk preparations were stirred for approximately 24 hours in the dark, then left to stand for approximately 24 hours in the dark. An aliquot (3 L) was then syphoned from a mid-vessel location of each preparation vessel, of which the first litre was discarded, to give 2 L aliquots of Water Accommodated Fractions (WAF) which were used as test media with nominal loading rates of 100, 31.3, 9.77, 3.05 and 0.95 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the log phase of growth.
- Method of cultivation: Liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth.

- Acclimation period: 6 days (pre-culturing)
- Culturing media and conditions (same as test or not): same
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Test temperature:
22.3-23.2 °C
test start (0 h): 7.98-8.03
test end (72 h): 8.00-8.67
Nominal and measured concentrations:
nominal loading rates: 0.95, 3.05, 9.77, 31.3 and 100 mg/L as WAF
measured levels: The measured levels of TOC were below the limit of quantification of the analysis method after background correction for the control medium.
Details on test conditions:
- Test vessel: 250 mL conical flasks (autoclaved) containing 100 mL of inoculated test medium and loosly plugged with foam bungs
- Aeration/gaseous exchange: orbital incubator, oscillating at nominal 130 cycles per minute
- Initial cells density: 1x10⁴ cells/mL
- Control end cells density: 150x10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes

- Source/preparation of dilution water: OECD medium according to guidelines, using filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 0.98 mg C/L
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 4440 to 8880 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts.

- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
not specified
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
69.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
31.3 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: results are expressed in terms of loading rates
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no biologically significant stimulation of growth was observed

At the start of the test, the test media were colourless.
Reported statistics and error estimates:
For determination of the ECx values the approaches recommended in the OECD guideline 201 were used. Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002), using nominal loading rates.
Validity criteria fulfilled:
The criteria of OECD Guideline 201 and EU Method C.3 for biomass and growth rates were fulfilled.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD 201, EU C.3):

EL50: > 100 mg/L, EL10: 69.1 mg/L

Key value for chemical safety assessment

Additional information

The results indicate that the test material is not inhibitory to growth of algae up to the limits of its water solubility (<1 mg/L) and a NOEC and thus a PNEC cannot be determined.