Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2018 - 06 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol

EC Number:

948-055-8

Molecular formula:

not applicable as it is a UVCB

IUPAC Name:

Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol

Test material form:

liquid: viscous

Details on test material:

- Physical appearance: dark brown to black viscous liquid
- Storage conditions: in refrigerator (2-8°C) protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from the limit concentration and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume: 4.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the test facility
Samples for determination for exposure concentrations were collected from an extra vessel incubated without microorganisms to avoid the presence of biological material and/or microorganisms in the collected volume.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:

- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying a 15 minute period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure, with the exception of the highest test concentration, which was slightly yellow.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. Cultures were max. 4 weeks old. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L

Test temperature:

19-20°C

pH:

Control: 8.0 (t=0 h) and 8.1 (t=48 h)
100 mg/L: 8.1 (t=0 h) and 7.9 (t=48 h)

Dissolved oxygen:

Control: 9.0 mg/L (t=0 h) and 9.1 mg/L (t=48 h)
100 mg/L: 9.6 mg/L (t=0 h) and 7.3 mg/L (t=48 h)

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration: at t=0: 92.9 mg/L; at t=48: 87.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, according to OECD 202
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: the test was performed in the dark

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, in the limit test concentration and the control. Temperature of the medium: continuously in a temperature control vessel.

COMBINED LIMIT/RANGE-FINDING STUDY: yes, twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and test concentrations of 0.10, 1.0, 10 and 100 mg/L. No immobility was observed and it was decided to perform the final test as limit test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (March 2018)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No immobility was observed in the control group or at the limit concentration in the 48 hours exposure period.
- No biological, behavioural or other abnormalities were observed in the control group or any of the test concentrations.
- Analyses of the samples taken from the nominal concentration of 100 mg/L showed a concentration of 92.2 mg/L at the start of the test. The measured concentration decreased to 94% of initial at the end of the test (87.7 mg/L). Based on this result, the effect concentration was based on the nominal limit concentration of 100 mg/L
- Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.33 mg/L, 95%-confidence interval: 0.29-0.38 mg/L
- Other: results fell within the historical range of the test facility.

Any other information on results incl. tables

Table 1 Analytical results of test samples

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	< 12^*	n.a.
	100	92.9	93
48	0	< 12^*	n.a.	n.a.
	100	87.7	88	94

^* A negative concentration was calculated, because the absorption was below the intercept of the calibration curve. Therefore the analyzed concentration is reported as < the lowest calibration solution (i.e. 12 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the control, no daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels (e.g., ≥ 7.3 mg/L).

Conclusions:

The 48h-EC50 of Cocoa Shell Extract (Pot) towards Daphnia magna, was determined to exceed the highest tested concentration (> 100 mg/L).