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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:
09 January 2018 - 16 August 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:

Test material

Constituent 1
Test material form:
semi-solid (amorphous): gel

Sampling and analysis

Analytical monitoring:
Details on sampling:
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 preliminary range-finding tests and initial experiments, Pseudokirchneriella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 0.0010, 0.0032, 0.010, 0.032 and 0.10 mg/L (three replicate flasks per concentration) 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.

Test solutions


Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

Study design

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

Test conditions

Test temperature:
24 ±1 ºC
Nominal and measured concentrations:
Based on the results of the further range-finding tests the following loading rates were assigned to the definitive test: 0.0010, 0.0032, 0.010, 0.032 and 0.10 mg/L. Due to the need to test at relatively low test concentrations, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the required test concentrations.
An initial test was terminated after 48 hours exposure due to possible contamination of the 0.0032 mg/L loading rate WAF test preparations.
Details on test conditions:
A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 20 liters of culture medium to give the 1.0 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 95 hours and the mixture allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions of test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate to give stock solutions of 0.10, 0.032, 0.010, 0.0032 and 0.0010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the WAFs was separately inoculated with algal suspension (2.0 mL) to give the required test concentrations of 0.0010, 0.0032, 0.010, 0.032 and 0.10 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

In the definitive test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each 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 1.10 x 106 cells per mL. Inoculation of 450 mL of test medium with 2.0 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 4000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):
A positive control (Envigo Study Number RD71YQ) used potassium dichromate as the reference item. The positive control was conducted between 06 November 2017 and 09 November 2017.

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
ca. 0.032 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
ca. 0.003 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Results with reference substance (positive control):
A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
Reported statistics and error estimates:
EL50 Growth Rate 0.065 - 0.085
EL 50 Yield 0.038 - 0.048

Applicant's summary and conclusion

Validity criteria fulfilled:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0026 to 0.061 mg/L except at the lowest test concentration which was less than the limit of quantification (LOQ) of the analytical method, determined to be 0.0014 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.0087 mg/L 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.

NOEL Growth Rate 0.032 mg/L

NOEL Yield 0.0032 mg/L
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella 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.