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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
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 202 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 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
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), at t=24h (new and old solutions) and at the end of the test (t=48h).
- Number of samples: Sampling will consist of duplicate 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 test loading rates. TOC analysis will not be performed in compliance with 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 (for fresh media at t=0h and t=24h) 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. 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 into test vessels (without headspace) or will be filtered if necessary. After filling and introduction of daphnids the test vessels will be sealed immediately. 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.

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

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Breeding conditions: Daphnids are cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids is performed in all-glass vessel containing test water. Cultures are maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids are fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water is changed at least once per week. These culture conditions allow to maintain the daphnids in the parthenogenetic reproductive stage.

Study design

Test type:
Water media type:
Total exposure duration:
48 h
Remarks on exposure duration:
Post exposure observation period:
No data

Test conditions

A water hardness between 140 and 250 mg.L-1 (as CaCO3) is recommended.
Test temperature:
Controlled environment cabinet (20°C ± 2°C); should not vary by more than 1°C at the end of the test)
Between 6.0-9.0. Should not vary by more than 1.5 units at the end of the test in any solution.
Dissolved oxygen:
Dissolved oxygen concentration at the end of the test should be ≥ 60% of the air-saturation value in controls and test vessels.
No data.
No data.
Nominal and measured concentrations:
Range-finding test on-going
Details on test conditions:
- Test duration: 48 hours
- Test type: Semi-static, in order to prevent the dissipation of the test item from test solutions because of its volatility and the expected degradation of some constituents.
- Test vessels: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Number of daphnids: 20 per treatment group (control and loading rate of 100 mg.L-1), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace
- Number of replicates: 4 replicates with daphnids per treatment group.
- Aeration: No aeration of the test solutions
- Feeding: No feeding
- Introduction of daphnids: Daphnids will be introduced into the test vessel each completely filled with test solution and without headspace as soon as possible after preparation of the test solutions.

- Test water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202.

- Photoperiod: 16 h light : 8 h dark

Immobility: Immobility and abnormal behaviour will be determined by visual observation after 24 and 48 hours. Immobile animals will be eliminated from the vessels as soon as they will be discovered. Daphnids are considered to be immobile if they are not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all treatment group.
- Temperature of medium: Measured continuously in a “temperature control” vessel next to the test vessels, over the study period, beginning at the start of the test.

- Defining exposure concentrations: Effective concentrations will be determined considering nominal WAF concentrations (nominal loading rate values).
- Estimation of EL50 after 24 and 48 hours of exposure (if applicable): The data will be analysed by regression to determine the EL50 including the 95 % confidence interval by using the computer program ToxRat. The highest loading rate without effect and lowest concentration with 100% immobilisation will be determined by visual observation. Optionally, other statistical analyses may be performed if appropriate.

- Preparation of test solutions: The range-finding test is carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rates of 1, 10, 32 and 100 mg.L-1 and to a control. Two method of preparation for the WAFs is tried: heating/sonication and solvent conditions. The results of this preliminary test will be used in order to determine the range of loading rates to be tested in the definitive test.
Reference substance (positive control):
Potassium dichromate

Results and discussion

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

Any other information on results incl. tables


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 acute toxicity of test item to Daphnia magna. The method followed is designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended byCommission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

The daphnids are exposed to Water Accommodated Fractions (WAFs) of the test item. The immobility of the daphnids is determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the control and the WAFs is checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

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