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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
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
The method was modified according to OECD series No. 23, to take the impact of the colored test item into account.
equivalent or similar to
EU Method C.3 (Algal Inhibition test)
Principles of method if other than guideline:
-Modification according to OECD series No. 23: Thee test item solutions were green-blue. Since this may affect the light intensities for the algae cultures, the volumes of test media in the test vessels were lowered in order to minimize the water layer and the path length. The light intensity was adjusted to the upper limit of the prescribed range.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Purity: 89% isomers of the tri- and tetra-sulphonated phthalocyanine zinc complex; 11% water
Analytical monitoring:
Details on sampling:
- Concentrations: at all treatments at the start (oh) and the end of exposure (72h)
- Sampling method: Sampies at the start of exposure were taken from an additional replicate without algae. At the end of exposure, test samples were taken directly from the test replicates.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- A stock solution of 100 mg/L was freshly prepared with dilution water. The stock solution was diluted to generate the different test concentrations.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Common name: Desmodesmus subspicatus CHODAT
- Strain: SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen.
- Age of inoculum (at test initiation): A four day old preculture, prepared in dilution water, was used as inoculum. For the start of the test, the preculture was directly pipetted into each test concentration and the control.

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Test temperature:
22.5 ± 0.5 °C
7.52 - 8.16
Nominal and measured concentrations:
Nominal concentrations: control, 1.00, 3.16, 10.0, 31.6 and 100 mg/L
Geometric mean measured concentrations: control, 0.389, 0.699, 1.28, 5.67 and 42.7 mg/L
Details on test conditions:
- Test vessel: Erlenmeyer flasks with cotton wool plugs
- Size, fill volume: 250 mL, 25 mL
- Initial cells density: 4332 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- other: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

- Source/preparation of dilution water: according to guideline (OECD 201 medium)
- Culture medium different from test medium: yes, nutrient medium Z (Z-agar)
- Intervals of water quality measurement:

- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 - 120 µE * m-2 * s-1, Fluorescent tubes, OSRAM L 36 W/11-865, cool daylight

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was counted daily in a plankton chamber.
- Chlorophyll measurement: The cell density of the preculture was measured via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm.
- Other: Algae cells were evaluated microscopically at the start and the end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

- Range finding study: yes
- Test concentrations: 10 and 100 mg/L (nominal)
Reference substance (positive control):
Potassium dichromate
Key result
72 h
Dose descriptor:
Effect conc.:
4.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
1.07 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- The measured concentrations of the substance at the start of exposure were in the range of 100 to 109 % of the nominal values.
- At the end of the exposure, recoveries were < LOQs to 18 %.
Results with reference substance (positive control):
The ErC50 (72h) of the control substance potassium dichromate was 0.631 mg/L.
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by linear regression. The calculation of the confidence intervals of EC10-, EC20- and EC50-values were carried from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

- The cell growth increased 206-fold from 0 to 72 hours in the control (required: at least 16 -fold).

- The mean coefficient of variation for section-by-section specific growth rates (days 0 -1, 1 -2 and 2 -3) in the control cultures was 27.3 % (required: < 35 %).

- Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.67 % (required: < 7 %).

- The temperature during the test was 22.5 ± 0.5 °C (required: 21 – 24 °C, within ± 2 °C).

- The pH-value in the control replicates increased by units from 8.03 to 8.16 (required: not more than 1.5 units).

Validity criteria fulfilled:
The test substance is toxic to aquatic algae.

Description of key information

Acutely toxic to aquatic algae.

EC50 (72h) = 4.57 mg/L and EC10 (72h) = 1.07 mg/L (nominal) for growth rate of Desmodesmus subspicatus (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
4.57 mg/L
EC10 or NOEC for freshwater algae:
1.07 mg/L

Additional information

The toxicity of ‘zinc phthalocyanine sulfonate’ on green alga was investigate in two guideline studies according to OECD 201 (BASF SE 2013, Procter & Gamble 1994). In both studies the substance shows strongly impacts on the growth of unicellular green algae. However, due to the fact that the test item solution is colored green-blue, the standard test procedure according to OECD guideline 201 is not on its own sufficient for testing the toxicity on algae. Modifications of the guideline test design are required, to exclude secondary effects on algae growth, based on reduced light intensity. Only one of the two available studies take this aspect into account (BASF SE 2013). Therefore, the second study (Procter & Gamble 1994) was excluded for the further hazard and risk assessment. 

In the reliable key study, the volumes of test media in the test vessels were lowered in order to minimize the water layer and the path length. The light intensity was adjusted to the upper limit of the prescribed range. This modification stands in accordance with the OECD series No. 23 (Guidance document on aquatic toxicity testing of difficult substances and mixtures, 2000). The study was performed according to OECD guideline 201 and GLP using the green algae Desmodesmus subspicatus as test organism. Test concentrations were monitored using spectral photometry. After 72 hours of exposure the 72h-EC50 is determined to be 4.57 mg/L and the EC10 is determined to be 1.07 mg/L based on measured concentrations (geometric mean).