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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 26, 2007-November 28, 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
equivalent or similar to guideline
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
according to guideline
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)

Test material

Details on test material:
- Physical state: Blue solid lumps
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: control and all concentrations
- Sampling method: 40 ml at t=0 h and t=48 h
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with loading rates of 10 and 100 mg/l. The aqueous mixtures were treated with ultrasonic waves for 15 minutes, followed by a 24-hour magnetic stirring period and a 3½ hour stabilisation period. The resulting mixture at 100 mg/l was clear and colourless but contained test substance particles, precipitate and a floating layer. The mixture at 10 mg/l was clear and colourless with a precipitate and a floating layer. Therefore, the mixtures were passed over glass wool, after which they were clear and colourless (very slightly blue at 100 mg/l).

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 10^4 cells/ml.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 continuously 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):Stock culture medium is M1 (according to the NPR 6505, formulated using Milli-Ro water); Pre-culture medium and test medium is M2, according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observations.

Test conditions

0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
The temperature of the test medium was 20.2°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.6 and 23.7°C. Temperature in the incubator remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
8.0 - 8.1
Nominal and measured concentrations:
Loading rate 0 mg/L t=0h: TOC = 0.8617 mg/L
Loading rate 0 mg/L t=48h: TOC = 0.5699 mg/L
Loading rate 100 mg/L t=0h: TOC = 0.8618 mg/L
Loading rate 100 mg/L t=48h: TOC = 0.4199 mg/L
The TOC contents in treated samples were comparable to those in control samples, indicating that the actual test substance concentration was very low. This is not unexpected considering the presence of precipitate and a floating layer in the solution, which were removed by passing the solution over glass wool.
Details on test conditions:
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Aeration:
- Initial cells density: An initial cell density of 1 x 10^4 cells/ml.
- Control end cells density: 56x 10^4 cells/ml.
- No. of vessels per concentration (replicates):
6 replicates of the control and the highest test concentration
3 replicates of the lower concentration
3 replicates of the highest test concentration and the control without algae
1 replicate of the lower test concentration without algae
2 replicates of the control without algae

- Standard medium used: yes

- Photoperiod: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 86 to 92 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
at 0. 24 and 48 h
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm).
- Spacing factor for test concentrations: 10 (0, 10 and 100 mg/L)
- Range finding study: the sudy is combined limit/range finding study
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrations
48 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): no abnormalities observed
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 2.1 mg/l with a 95 % confidence interval ranging from 1.5 to 3.0 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

Applicant's summary and conclusion

Validity criteria fulfilled:
No reduction of growth rate or inhibition of yield was recorded at any of the concentrations of the test substance.

The NOEC for growth rate reduction and yield inhibition was 100 mg/l based on loading rates.

Due to the very low solubility of the test substance in water, concentration levels that might be toxic for algae could not be reached. Hence the 48h-EC50 for both growth rate reduction and yield inhibition was above a loading rate of 100 mg/l.

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