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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-09 to 2018-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is performed according to the OECD 201 guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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:

Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test. 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 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 22.5 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 added into test flasks containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the test solutions were observed to be clear and slightly coloured (yellowish/light-brown) mostly at the highest loading rates. The test was carried out without adjustment of the pH.

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

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

23°C ± 2°C

pH:

Should not vary by more than 1.5 units at the end of the test in the control.

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Range-finding test on-going

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test vessels: Test vessels 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace. Each test vessel was 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 were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were 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 were prepared for chemical analyses.

TEST WATER
- 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

EFFECT PARAMETERS MEASURED :
- Cell densities: Cell numbers were 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.

DATA HANDLING
- Defining exposure concentrations: nominal loading values of 1.0, 3.2, 10.0, 32.0 and 100.0 mg.L-1 and to a control
- 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.

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; 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):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

88.238 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CF: 71.724-115.582

Details on results:

The 72-hour EL50 for the parameters growth rate and yield were determined to be 88.2 mg.L-1 and 16.2 mg.L-1, respectively. The 72-hour EL10 for growth rate was 9.7 mg L-1 and for yield was 6.6 mg.L-1.

Results with reference substance (positive control):

72h-EC50 was 0.98 mg.L-1 for the parameter growth rate.

Reported statistics and error estimates:

95% conf. limits 71.724 – 115.582

Validity criteria of the study:

Cell density in controls 239-fold increase within 72 hours. The specific growth rate was determined at 1.82 day-1, so greater than 0.92 day-1.

Coefficient of variation 1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 9.0%. 2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 2.7%.

Physico-chemical parameter values throughout the test

Although the pH level in the control varied by more than 1.5 units at the end of the test (2.32 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of more sodium bicarbonate. The mean light intensity was of 5252 lux (range: 4703-5710 lux), which remained within the ranges prescribed by the study plan (range: 4440-8880 lux and shall not vary more than ± 15% from the average light

intensity over the incubation area). Temperatures were situated between 23.6 and 25.2°C throughout the test (average value: 23.8°C), which was slightly higher than the requirements laid down in the study plan (23°C ± 2°C, constant within 2°C). However, this minor deviation was considered not to affect the results of the test as no impact was observed on controls throughout the duration of the test.

pH-values during the final test

Nominal concentration (mg test item.L-1)*
	Control	1	3.2	10	32	100
Start t=0h	8.2	8.2	8.17	8.08	8.1	7.94
End t=72h	10.52	10.33	10.1	9.89	7.8	7.57

Analytical results

Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and at the end of the test.

Although every effort was made to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOC analyses indicated that organic compounds in WAFs were maintained between 14 and 39% of the initial TOC concentration between the start and the end of the test, except for the loading rates of 1.0 mg.L-1 with a gain of 65%. It would be difficult to determine the specific cause of this last observation (measurement uncertainty of the TOC analyser). This probable overestimation constituted a deviation from the test guideline (higher than ± 20% of the initial concentrations values) but was considered not to affect the results and the integrity of the study regarding properties of the test item and the non-specific analytical method.

However, despite these analytical results, the study was considered as valid given the results from the range-finding test which are consistent with the final test outcome, in agreement with the Study Monitor.

Moreover, it should be noted that the study was carried out using WAFs of a natural complex substance made of several constituents with different stabilities, solubilities and behaviours in aqueous solutions during testing.

Therefore, 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	End t=72h	Relative loss to initial value (t=0h - t=72h) (%)
Control	0.92	0.34	63
1	1.22	2.01	-65
3.2	2.05	1.52	26
10	4.47	2.71	39
32	13.6	11.7	14
100	38.4	24.4	36
* WAF prepared at the given loading rate. N.A.: not applicable

Biological results

Algal cell densities during the final test (expressed as density of algal cells.mL-1x10⁴).

	Replicate	Nominal concentration (mg test item.L-1)*
		Control	1	3.2	10	32	100
t=24h	1	6.8	8.8	6.4	8	4.8	1.6
	2	7.6	6	7.2	7.6	4	2
	3	8.4	6.8	8	7.2	4	1.6
	4	8.4
	5	8
	6	6.8
	Mean	7.7	7.2	7.2	7.6	4.3	1.7
	Std.Dev.	0.73	1.44	0.8	0.4	0.46	0.23
	%Inh.	-	2.6	2.3	0.2	21.5	54.6
t=48h	1	42.4	38.4	30	35.6	9.2	4
	2	38.8	37.2	37.2	43.2	8	3.2
	3	44	39.2	39.6	36.4	9.2	3.2
	4	43.2
	5	44
	6	40.8
	Mean	42.2	38.3	35.6	38.4	8.8	3.5
	Std.Dev.	2.05	1.01	5	4.18	0.69	0.46
	%Inh.	-	2.2	4	2.2	35.4	56.5
t=72h	1	150	106	104	84	20	8.8
	2	132	130	99.2	114	15.6	8
	3	112	116	120	80	19.2	6.4
	4	116
	5	98
	6	110
	Mean	119.7	117.3	107.7	92.7	18.3	7.7
	Std.Dev.	18.48	12.06	10.89	18.58	2.34	1.22
	%Inh.	-	0.2	1.8	4.7	34.3	50.1

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EL50 for the parameters growth rate and yield were determined to be 88.2 mg.L-1 and 16.2 mg.L-1, respectively. The 72-hour EL10 for growth rate was 9.7 mg L-1 and for yield was 6.6 mg.L-1.

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 start and the end of the test.

The 72-hour EL50 for the parameters growth rate and yield were determined to be 88.2 mg.L-1 and 16.2 mg.L-1, respectively. The 72-hour EL10 for growth rate was 9.7 mg L-1 and for yield was 6.6 mg.L-1.

Description of key information

The 72-hour EL50 for the parameters growth rate and yield were determined to be 88.2 mg.L-1 and 16.2 mg.L-1, respectively. The 72-hour EL10 for growth rate was 9.7 mg L-1 and for yield was 6.6 mg.L-1.

Key value for chemical safety assessment

EC50 for freshwater algae:

88.2 mg/L

Additional information

The toxic effect of the test item Cacao Oleoresin (Theobroma cacao) to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated

Fractions. Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 88.2 mg.L-1 and 16.2 mg.L-1, respectively. The 72-hour EL10 for growth rate was 9.7 mg L-1 and for yield was 6.6 mg.L-1.