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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006; Annex 5 corrected 28 July 2011

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:

Samples for possible analysis were taken from all test concentrations and the control:
- Frequency: at t=0 h, t=24 h, and t=72 h
- Sampling volume: 4.0 mL; at the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

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 the highest test concentration, which was slightly yellow.
- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 d (pre-culture)
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C, until the pre-culture started.

PRE-CULTURE:
- Pre-culture method: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2; according to the OECD 201 Guideline, formulated using Milli-RO water) at a cell density of 1 x 10^4 cells/mL.
- Culturing media and conditions (same as test or not): Yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO2/L

Test temperature:

22-24°C

pH:

At the start of the test: 8.0
At the end of the test: 8.2-8.6

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100 mg/L
Measured concentrations: at the end of the test: 7.9, 15.3, 30.7, 55.9 and 96.7 (100-115% of initial). For more details, see table 1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, capped vessels
- Test solution volume: 50 mL
- Aeration: No
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: 238.6x10^4 (stdev: 6.2x10^4)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 2 extra replicates without algae of each test group for sampling purposes and 1 replicate of each concentration without algae


GROWTH MEDIUM
- Standard medium used: yes (M2, according to OECD 201 Guideline)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 test medium, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Stock culture medium: M1, pre-culture medium: M2.
- Intervals of water quality measurement: pH at the beginning and at the end of the test; Temperature of medium continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 96 to 99 µE.m^2.s^1.
- During incubation the algal cells were kept in suspension by continuous shaking.


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and acounting chamber; thereafter cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the 32 mg/L concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Six replicates of exponentially growing algae were exposed to a control and test concentrations of 0.10, 1.0, 10 and 100 mg/L. At a concentration of 100 mg/L growth rates and yield were inhibited by 39 and 88%, respectively. Based on these results, the concentrations for the final test were determined.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate; March 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

A dose-related increase in inhibition of growth rate was observed with percentages of inhibition of 12, 23 and 40% at test concentrations of 32, 56 and 100 mg/L, respectively. See table 2 for details.

- Exponential growth in the control: yes
- Observation of abnormalities: no

- Results of analytical determination of test concentrations: Measured concentrations were at the level of nominal throughout the test, except at the lowest test concentration, where the concentrations were at the level of 78-79% of nominal. However, since the criterion of ±20% variation from nominal was only slightly exceeded, and biological results at the lowest test concentration were not crucial for determination of effect parameters, it was decided to base the effect parameters on analytically confirmed nominal concentrations. It should be noted that a small response was observed in the control. However, since a similar response was observed in the quality control samples, it was most probably due to carry over.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: The EC50 for growth rate inhibition (72h-ErC50) was 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.6 mg/L.
- The results were within the historical range of the test facility.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

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	< 8^*	n.a.
	10	7.9^#	79
	18	14.4	80
	32	28.0	88
	56	48.5	87
	100	89.6	90
24	0	1.8^#	n.a.	n.a.
	10	7.8^#	78	99
	18	14.8	82	103
	32	28.7	90	102
	56	51.8	92	107
	100	95.1	95	106
72	0	1.2^#	n.a.	n.a.
	10	7.9^#	79	100
	18	15.3	85	106
	32	30.7	96	110
	56	55.9	100	115
	100	96.7	97	108

* 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. 8 mg/L).

# Estimated value, calculated by extrapolation of the calibration curve.

Table 2 Growth rate and percentage inhibition for the total test period

Cocoa Shell Extract (Pot) Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.825	0.0085	6
10	1.818	0.0058	3	0.4
18	1.790	0.0164	3	1.9
32	1.611	0.0273	3	12*
56	1.413	0.0232	3	23*
100	1.102	0.0514	3	40*

* - effect was statistically significant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks'

Conclusions:

The 72h-ErC50 of Cocoa Shell Extract (Pot) towards aquatic algae was determined to exceed the highest tested concentration (> 100 mg/L). The 72h-NOEC and the 72h-EC10 for growth rate inhibition were 18 and 32 mg/L, respectively.