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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:
9 February - 26 March 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The 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
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): Aquapel® 203
- Substance type: yellow fatty liquid
- Physical state: liquid
- Analytical purity: 88.7%
- Lot/batch No.: G11AN059
- Expiration date of the lot/batch: 21 January 2010
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: A WAF prepared at a loading rate of 100 mg/l
- Sampling method: Duplicate samples for possible analysis were taken from both test groups according to the following schedule:
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2 ml

- Sample storage conditions before analysis:Not applicable, samples were analysed on the day of sampling.

Test solutions

Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/l applying 1 day of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixture was left to stabilize for 1 day after which the slightly hazy Water Accommodated Fraction (WAF) was taken out. Part of the WAF was used for pre-incubation of the test vessels (1½ hour). The remaining part of the WAF was used for testing.

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): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) 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.

Culturing media and conditions (same as test or not): Same as test

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:

Test conditions

24 mg CaCO3/l
Test temperature:
22.1 and 23.1°C
7.6 - 8.5
Dissolved oxygen:
Not measured.
Not applicable.
Nominal and measured concentrations:
At the start of the test, the mean actual test concentration in the WAF prepared at a loading rate of 100 mg/l was 9.3 mg/l. This concentration decreased to 4.9 mg/l after 24 hours of exposure and further to 1.4 mg/l at the end of the test. Based on these results, the Time Weighed Average exposure concentration (TWA) was calculated to be 4.0 mg/l.

Details on test conditions:
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 ml, all-glass, containing 50 ml of test solution
- Aeration: During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/ml.
- Control end cells density: 140 x 10^4 cells/ml.
- No. of organisms per vessel: 1 x 10^4 cells/ml.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

- Standard medium used: yes

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 and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.2

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24h
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 77 to 82 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The test solutions were so turbid that they disturbed spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test.

Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
> 4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
< 4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Details on results:
No significant differences were recorded between the values for growth rate reduction at any of the test concentrations when compared to the control group.
A TWA concentration of 4.0 mg Aquapel® 203 per litre induced a statistically significant inhibition of yield of 13% (2 Sample t-Test, α = 0.05).

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: Under the conditions of the reference study with Pseudokirchneriella subcapitata, potassium dichromate reduced growth rate of this fresh water algae species at 0.56 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.6 mg/l with a 95 % confidence interval ranging from 1.2 to 2.1 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.

Reported statistics and error estimates:
A TWA concentration of 4.0 mg Aquapel® 203 per litre induced a statistically significant inhibition of yield of 13% (2 Sample t-Test, α = 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
Due to the very low solubility of Aquapel® 203 in test medium, concentration levels that might induce 50% or more effect on algal growth could not be reached.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) was above 4.0 mg/l, which was above the given water solubility (< 1 mg/l).

The NOEC for growth rate reduction was 4.0 mg/l and the NOEC for yield inhibition was < 4.0 mg/l.