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Diss Factsheets

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:
02 August 2012 to 14 September 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This 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)
Version / remarks:
according to guideline
EU Method C.3 (Algal Inhibition test)
Version / remarks:
according to guideline
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
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:

Test material

Constituent 1
Reference substance name:
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PAZ-D
- Molecular formula: C43H50O4N8F2
- Molecular weight: 780.9
- Physical state: white powder
- - Expiration date of the lot/batch: 13 January 2013 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- Water solubility: 0.0453 mg/L
- Stability in water: No

Sampling and analysis

Analytical monitoring:
Details on sampling:
Duplicate samples for possible analysis were taken from the limit concentration and the control according to the schedule below.

Frequency: at t=0 h and t=24 h
Volume: 0.4 mL
Storage : Not applicable, samples were analysed on the day of sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and after 24 hours of exposure.

Test solutions

Details on test solutions:
The batch of PAZ-D tested was a white powder with a purity of 99.3% and not completely soluble in test medium at the loading rate initially prepared.

Preparation started with a loading rate of 100 mg/L applying an overnight period of magnetic stirring. Thereafter, the obtained mixture was filtered through a 0.45 µm membrane filter (rs 55, Whatman) and used as the highest test concentration. The lower test concentrations in the range-finding test were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

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.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Source: In-house laboratory culture.

Stock culture: 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.

Pre-culture: 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 10e4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

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

Test conditions

0.24 mmol(24 mg CaCO3/L)
Test temperature:
The temperature of the test medium was 20.5°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.2 and 22.5°C.
8.0 - 8.5
Dissolved oxygen:
not measured
not measured
Nominal and measured concentrations:
Nominal: 0.45 µm filtered solution prepared at a loading rate of 100 mg/L
Average exposure concentraton: 0.64 ug/L

Due to possible adsorption to glassware the obtained analytical results should be considered as estimated values.
Details on test conditions:
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 123.7 x 10e4 cells/ml
6 replicates each for the control and the limit concentration.
1 replicate of the highest concentration without algae.
1 or 2 replicates for sampling purposes.

- Standard medium used: yes
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 6767 to 7194 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h ErC50,

- Test concentrations: control and 0.45 um filtered solution prepared at 100 mg/l
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
> 0.64 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: -
72 h
Dose descriptor:
Effect conc.:
0.64 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: -
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 2.3 mg/l
Reported statistics and error estimates:
Determination of the average exposure concentrations
The average exposure concentrations were calculated as , being the geometric means of the concentrations of PAZ-D measured in the samples taken at the start (Ct=0) and after 24 hours of exposure (Ct=24).

In case concentrations measured are below the limit of detection or the limit of quantification, the final exposure concentration(s) will be taken as a factor of 2 below the applicable limit. This procedure is based on the OECD “Guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment”.

Determination of the NOEC and calculation of the EC50
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used.

Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).

No EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration).

Applicant's summary and conclusion

Validity criteria fulfilled:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at the average exposure concentration of 0.64 µg PAZ-D/L.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) were beyond the range tested, exceeded the average exposure concentration of 0.64 µg/L.

The NOEC for both growth rate reduction and yield inhibition was 0.64 µg/L.

Due to the very low solubility of PAZ-D in water, concentration levels that might be toxic for algae could not be reached.