Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with ethanol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-16 to 2018-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is performed according to the OECD 202 guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

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: 4 replicates with daphnids per treatment group.
- Analytical monitoring: Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were
immediately sealed with screw caps after weighing. Each glass flask was 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 extract the soluble fraction of the test item as much as possible.
Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were filtered and added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear. The test was carried out without adjustment of the pH.
Test item: loading rates (spaced by a factor of 1.35): 30, 41, 55, 74 and 100.0 mg.L-1.
Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

No data

Hardness:

approximately 250 mg.L-1 (as CaCO3).

Test temperature:

between 20.8 and 20.9°C

pH:

Between 6.0-9.0.

Dissolved oxygen:

The dissolved oxygen concentration was ≥ 3 mg.L-1 throughout the test in all test vessels

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal loading values of 30, 41, 55, 74 and 100.0 mg.L-1

Details on test conditions:

TEST PROCEDURE AND 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 were introduced into the test vessels each completely filled with test solution and with minimum headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Test water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED
Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were 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 controlled vessel next to the test vessels, over the study period, beginning at the start of the test.


RANGE FINDING TEST
- The range-finding test was 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 (acetone); heating/sonication). The WAFs (for fresh media at t=0h and t=24h) were prepared in the dark under closed conditions and by slow-stirring.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Details on results:

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the tested loading rates, suggesting that immobilisation of daphnids observed during the range-finding test at 100 mg.L-1 was probably due to a lack of dissolved O2.
Based on these results, the 24 and 48-hour EL50 values were therefore > 100 mg.L-1.

Results with reference substance (positive control):

the 24h-EC50 was 1.31 mg.L-1.

Reported statistics and error estimates:

not relevant

Validity criteria of the study

Controls In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [O2] The dissolved oxygen concentration was ≥ 3 mg.L-1 throughout the test in all test vessels, except at the loading rates of 74 and 100 mg.L-1 at t=48h. This deviation was not considered to

have affected the results and the integrity of the study since no immobilisation was observed at any of the tested loading rates, in agreement with the Study Monitor.

Thus the validity criteria have been fulfilled in the present study.

Water quality parameters and environmental conditions throughout the test

These parameters remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: 3 mg.L-1 at the end of the test, except at 74 and 100 mg.L-1 (see 3.1.)), suggesting that pH and oxygen concentrations were influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.8 and 20.9°C throughout the test (average value: 20.8°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

pH-values during the final test.

Nominal concentration(mg test item.L-1)*
	Control	30	41	55	74	100
Start t=0h	8.07	7.36	7.28	7.17	7.08	7.01
t=24h Old	8.11	7.39	7.24	7.11	7.07	7
t=24hFresh	8.01	7.5	7.52	7.39	7.33	7.18
End t=48h	8.06	7.07	7.03	7	6.96	6.7

Dissolved oxygen concentrations (mg.L-1) during the final test.

Nominal concentration(mg test item.L-1)*
	Control	30	41	55	74	100
Start t=0h	8.3	6.6	6.37	5.7	5.2	4.54
t=24h Old	8.27	6.04	5.75	4.75	3.7	3.24
t=24hFresh	8.3	7.55	7.1	7	6.6	5.85
End t=48h	8.35	5.3	4.4	3.35	2.8	2.3

Analytical results

Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). TOC analyses indicate that organic compounds in the loading rates were stable the first day of the test (within the ± 20% of the initial TOC concentrations values), and less stable the second day (losses of approx. 35-45%). However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. In any case, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on nominal loading rates.

Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L-1) - Final test.

Nominal concentration* (mg test item.L-1)	Start (t=0h) t	t=24h Old	Relative loss to initial value (t=0h - t=24hold) (%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (t=24hfresh - t=48h) (%)
Control	1.2	0.9	25	1.3	0.7	46
30	10.9	8.9	18	9.1	5.3	42
41	14.4	15	-4	16.3	8.9	45
55	17.1	17.6	-3	18.8	9.6	49
74	24.2	24	1	23.6	15.9	33
100	31.2	29.1	7	31	20.5	34

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 was determined to be higher than 100 mg.L-1 (limit test).

Executive summary:

A study was performed to assess the acute toxicity of the test item Cacao Oleoresin (Theobroma cacao) to Daphnia magna. The followed method was 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 by Commission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 30, 41, 55, 74 and 100.0 mg.L-1 and to a control. The immobilisation of the daphnids was 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 was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

TOC analyses indicate that organic compounds in the loading rates were stable the first day of the test (within the ± 20% of the initial TOC concentrations values), and less stable the second day (losses of approx. 35-45%). However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the tested loading rates.

The toxic effect of the test item Cacao Oleoresin (Theobroma cacao) to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 was determined to be higher than 100 mg.L-1. Moreover, the highest nominal loading rate without observed effects value was determined to be at least 100 mg.L-1.

Description of key information

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 was determined to be higher than 100 mg.L-1 (limit test).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A reliable experimental study was available on the registered substance and was performed under GLP compliance.

The study was performed to assess the acute toxicity of the registered substance to Daphnia magna and was investigated in a closed semi-static test using Water Accommodated Fractions. The method followed was designed to be compliant with the OECD 202 Guideline and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 30, 41, 55, 74 and 100.0 mg.L-1 and to a control. The immobilisation of the daphnids was 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 was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

TOC analyses indicate that organic compounds in the loading rates were stable the first day of the test (within the ± 20% of the initial TOC concentrations values), and less stable the second day (losses of approx. 35-45%). However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the tested loading rates.

All validity criteria were fulfilled and the study respected the requirements of the guideline. This study is therefore considered acceptable for that endpoint.