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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:
From 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
This study is performed according to the OECD 201 guideline.
Justification for type of information:
Study on-going.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
Principles of method if other than guideline:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with water and ethanol
EC Number:
Molecular formula:
not applicable
Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with water and ethanol
Specific details on test material used for the study:

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Frequency of sampling: At start (t=0h) and at the end of the test (t=72h).
- Concentrations: Samples for analysis will be taken from all test concentrations (WAFs) and control.
- Number of samples: Sampling will consist of single samples taken per treatment.
- Analytical monitoring: Concentration of dissolved organic material in the WAFs will be checked by analysis of Total Organic Carbon (TOC) in the control medium and the loading rates. TOC analysis will not be performed in compliance with the OECD GLP principles but will be adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Test solutions

Details on test solutions:
The study will be carried out using WAFs (Water Accommodated Fractions). The WAFs will be prepared under closed conditions and by slow-stirring.
The mixing vessels will be 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar will be placed in each mixing vessel completely filled with test water (with a minimum headspace). The loading rates of the test item will be weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and will be immediately sealed with screw caps after weighing. Each glass flask will be placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to enhance the extraction of the soluble fraction of the test item as much as possible.
Then the mixing vessels will be carefully filled with the contents of the glass flasks and thereafter will be closed immediately. The mixing will be initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 +/- 2 hours of gentle stirring in the dark at room temperature, the WAFs will be allowed to stand for at least 1 hour before use. The first 100 mL will be discarded via the drain port. Then the WAFs will be directly added (or will be filtered if necessary) into test flasks containing a fixed amount of inoculum (5.10^3 cells.mL-1 per vessel) that will be immediately sealed after filling with a minimum headspace. The test will be carried out without adjustment of the pH. Depending on the behaviour of the test substance in test water, minor changes in the method of preparation may be carried out and will be reported in the study report.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Species: Pseudokirchneriella subcapitata, CCAP 278/4
- Origin: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Reason for selection: This system is a unicellular algal species susceptible to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- Stock culture: Algae stock cultures are started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
- Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture are inoculated in test water at a cell density of 1.10^4 cells.mL-1. The pre-culture is maintained under the same conditions as used in the test. The cell density is measured immediately before use.

Study design

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

Test conditions

No data
Test temperature:
23°C ± 2°C
Should not vary by more than 1.5 units at the end of the test in the control.
Dissolved oxygen:
No data.
No data.
No data.
Nominal and measured concentrations:
Range-finding test on-going
Details on test conditions:
- Test vessels: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Cell concentration: An initial cell density of 5.10^3 using an exponentially growing pre-culture.
- Test environment: Controlled environment cabinet (23°C ± 2°C); vessels will be distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells will be kept in suspension by continuous magnetic stirring.
- Light regime/intensity: Continuous illumination using cool white light with a light intensity of 60-120 μE.m-2.s-1 (equivalent range: 4440-8880 lux) and shall not vary more than ± 15% from the average light intensity over the incubation area.
- Replicates: 6 controls and 3 replicates of each loading rate for counting. Moreover, replicates of each treatment will be prepared for chemical analyses.

- Standard medium used: yes: Original medium of OECD TG 201, Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1

- Cell densities: Cell numbers will be counted daily by microscope using a counting chamber.
- pH: At the start and the end of the test in at least one vessel per treatment. The pH of the solution should preferably not deviate by more than 1.5 units during the test.
- Temperature of medium: Measured continuously in the growth chamber.
- Light intensity: Light intensity will be measured once (t=0h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

- Defining exposure concentrations: Effective concentrations will be determined considering nominal WAF concentrations (nominal loading rate values).
- Determination of algal growth inhibition (if applicable): average growth rate and yield inhibition
- Statistical analysis: The software used to perform the statistical analysis will be ToxRat® Professional. Optionally, other statistical analyses may be performed if appropriate. EL10, EL20 and EL50 will be calculated (including 95% confidence interval) when possible for both growth rate and yield. When possible, LOELR and NOELR will be determined for both growth rate and yield.

The range-finding test is carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg.L-1 and to a control, according to two methods of preparation: use of solvent; and heating/sonication. The results of this preliminary test will be used to determine the range of loading rates to be used in the definitive study.
Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrations
72 h
Dose descriptor:
Nominal / measured:
Conc. based on:
test mat.
loading rate of WAF
Basis for effect:
growth rate
Remarks on result:
not determinable
No results available since the study is on-going
Details on results:
No results available since the study is on-going
Results with reference substance (positive control):
No results available since the study is on-going
Reported statistics and error estimates:
No data since the study is on-going

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
No results are available at the moment because the study is on-going.
Executive summary:

A study is performed under GLP conditions to assess the test item for its ability to generate toxic effects on the unicellular algal species Pseudokirchneriella subcapitata. The method followed is designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Algal cells are exposed to Water Accommodated Fractions (WAFs) of the test item. The potential inhibition of growth in relation to control cultures is determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the control and the test loading rates is checked by TOC analysis at the startand the end of the test.

No results are available at the moment because the study is on-going.