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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:
24-nov-2008 -- 18-dec-2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or 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)
according to guideline
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
according to guideline
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid
EC Number:
Cas Number:
Molecular formula:
C17 H16 O5
2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid
Details on test material:
- Name of test material (as cited in study report): ASA404 C7
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 072301
- Expiration date of the lot/batch: 18 March 2009
- 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: all test concentrations and control
- Sampling method: volume 2 ml, at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer 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 a loading rate of 100 mg/l applying a 10-minute treatment period with ultrasonic waves followed by 49 hours of magnetic stirring to accelerate the dissolving of the test substance in the test medium. Obtained solutions contained undissolved material dispersed through and floating on the surface. The dispersion was consequently filtered through a 0.45 µm membrane filter (Whatman, RC55) to remove the non-dissolved material. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. . The final test solutions were all clear and colourless. Note that the control received similar treatment to prevent for any possible treatment related effects.

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 104 cells/ml.

- Controls: blank control and reference substance

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 at a cell density of 1 x 104 cells/ml.
- 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.

Study design

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

Test conditions

0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
22.0 - 23.7°C.
At t=0 h: 7.9 - 8.1
At t=72 h: 6.1 - 8.3
Nominal and measured concentrations:
Nominal concentrations: 0.1, 1.0, 10 and 100 mg/l
Measured concentrations:
At t=0 h: 99% of nominal
At t=72 h: 106% of initial
Details on test conditions:
- Test vessel: 100 ml, all-glass,
- Type: open
- Material, size, headspace, fill volume: glass, 150 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: ca 257000 cells/m;.
- No. of vessels per concentration (replicates): 6 replicates of the highest concentration, 3 replicates for in-between concentrations.
- No. of vessels per control (replicates): 1 replicate of each test concentration without algae.
- No. of vessels per vehicle control (replicates): 6

- Standard medium used: yes

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: 60 to 120 µE/m2/s using TLD-lamps of the type ‘Cool-white’ of 30 Watt

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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). Algal medium was used as blank and the extra replicates as background for the treated solutions.
72 h NOErC, 72 h NOEbC, 72 H ErC50, 72 h EbC50

- Spacing factor for test concentrations: 10
- Test concentrations: 0.1, 1.0, 10 and 100% of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l
Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrations
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50:
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.7 mg/l with a 95 % confidence interval ranging from 1.2 to 2.5 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.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.58 mg/l with a 95 % confidence interval ranging from 0.36 to 0.92 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:
It is concluded that this test is valid and that Pseudokirchneriella subcapitata, ASA404 C7 did not reduce growth rate at any of the concentrations tested. The yield was significantly reduced at 100 mg/l.

The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded 100 mg/l.

The NOEC for growth rate was 100 mg/l.

The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded 100 mg/l.

The NOEC for growth rate was 100 mg/l.

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