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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
Study period:
18 January 2018 - 21 February 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
not specified
according to guideline
EU Method C.3 (Algal Inhibition test)
not specified
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch: 52611021
Purity: 95 to 100%
Physical state/Appearance: clear yellow liquid
Analytical monitoring:
Details on sampling:
- Concentrations: single loading rate of 100 mg/L
- Sampling method: Samples were taken at 23, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
culture medium
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances) :

A nominal amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through two pieces of filter paper was required to ensure all dispersed test item was removed. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test item present.
An aliquot (1 liter) of the 100 mg/L loading rate WAF was inoculated with algal suspension (7.2 mL) to give the required test concentration of 100 mg/L loading rate WAF.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: The master cultures were maintained in the laboratory under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

- Culturing media and conditions: same as test
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
not specified
not specified
Test temperature:
maintained at 24 ±1 ºC throughout the test.
7.5 - 8.4
Dissolved oxygen:
not specified
not specified
not specified
Nominal and measured concentrations:
Range-finding Test: 10 and 100 mg/L loading rate WAF.
Definitive Test: 100 mg/L loading rate WAF
Details on test conditions:
As in the range-finding test 250 mL glass conical flasks were used.
Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.91 x 105 cells per mL. Inoculation of 1 liter of test medium with 7.2 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Reference substance (positive control):
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
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 abnormalities
- Unusual cell shape: no
- Colour differences: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.6 mg/L;
- Other: No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Validity criteria fulfilled:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The NOEL was 100 mg/L loading rate WAF.

Description of key information


A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.


Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a WAF of the test item, at a single nominal loading rate of100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.


Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured test concentrations of 0.12 and 0.042 mg/L respectively were obtained indicating that the test item was unstable under the conditions of the test.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitatato the test item gave EL50values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Additional information