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

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Feb 2018 - 29 Mar 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

17 July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

1995

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

Specific details on test material used for the study:

The test item is stable in water.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: Not applicable, freshly obtained sludge was used immediately.
- Concentration of suspended solids: 3.2 g/L in the concentrated sludge, 9.6 mg suspended solids/L in the test vessels.
- Preparation of inoculum for exposure: The sludge was washed with mineral medium.
- Pretreatment: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

12 mg/L

Based on:

TOC

Initial test substance concentration 2

Initial conc.:

28 mg/L

Based on:

test mat.

Initial test substance concentration 3

Initial conc.:

1.6 other: mg CO2/mg

Based on:

ThCO2

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONCENTRATIONS:
- Test solutions: bottle A: 27.52 mg test item per litre; bottle B: 27.56 mg test item per litre

CONTROLS:
- Inoculum blank: containing only inoculum (2 bottles)
- Positive control: containing reference item and inoculum (1 bottle).
- Toxicity control: containing test item, reference item and inoculum (1 bottle).

PREPARATION OF TEST SOLUTIONS:
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. TOC content of the test item was determined by TOC analysis to be 43.65%. On the day of testing weighed amounts of the test item were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.

TEST CONDITIONS
- Composition of medium: According to OECD 301B
- Test temperature: 22-23 °C
- pH: at start: 7.6; t=14d: 7.8 (positive and toxicity control); t=28d: 7.4-7.5 (blank controls and test solutions)
- pH adjusted: yes, the pH was adjusted in all vessels to 7.6 using 1 M HCl.
- Suspended solids concentration: 9.6 mg suspended solids/L
- Test media were excluded from light

TEST SYSTEM
- Number of culture flasks/concentration: 2 for test suspension, 2 for inoculum blank, 1 for positive control, 1 for toxicity control.
- Method used to create aerobic conditions: synthetic air (CO2 < 1 ppm) sparged through scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Details of trap for CO2 and volatile organics: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.
- Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.

SAMPLING
- Sampling frequency: Titrations were made on day 2, 5, 8, 12, 15, 19, 23 and 29. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Titration of Ba(OH)2 in the gas scrubbing bottles.

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.5%; ThCO2: 1.07 mg CO2/mg

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (CO2 evolution)

Value:

73

Sampling time:

28 d

Remarks on result:

other: Bottle A

% Degradation 2

Key result

Parameter:

% degradation (CO2 evolution)

Value:

71

Sampling time:

28 d

Remarks on result:

other: Bottle B

Details on results:

- The ThCO2 of Cocoa Shell Extract (Pot) was calculated to be 1.60 mg CO2/mg.
- Biodegradation: The relative biodegradation revealed 73% and 71% biodegradation of the test item (based on ThCO2), for the duplicate bottles tested. The criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was met.
- Toxicity control: More than 25% biodegradation occurred within 14 days (70%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:

The positive control, sodium acetate, showed a normal biodegradation curve and reached 84% biodegradation after 14 days, which passes the validity criterium of at least 60% biodegradation within 14 days.

Any other information on results incl. tables

Table 1 CO₂ Production and Percentage Biodegradation of the Test Item (Bottle A)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^*)(%)
	Blank (mean)	Bottle A
2	46.16	31.76	14.40	15.8	15.8	18
5	43.62	23.26	20.36	22.4	38.2	43
8	41.83	32.42	9.41	10.3	48.6	55
12	42.26	36.66	5.60	6.2	54.7	62
15	43.56	40.96	2.60	2.9	57.6	65
19	43.58	41.59	1.99	2.2	59.8	68
23	44.00	42.00	2.00	2.2	62.0	70
29^#)	44.30	43.20	1.10	1.2	63.2	72
29^#)	47.75	47.08	0.67	0.7	63.9	73
29^#)	49.55	49.06	0.48	0.5	64.5	73
^*): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.0 mg CO2/2L. ^#): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

Table 2 CO₂ Production and Percentage Biodegradation of the Test Item (Bottle B)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^*)(%)
	Blank (mean)	Bottle B
2	46.16	36.90	9.26	10.2	10.2	12
5	43.62	22.69	20.93	23.0	33.2	38
8	41.83	30.94	10.89	12.0	45.2	51
12	42.26	34.23	8.03	8.8	54.0	61
15	43.56	40.56	3.00	3.3	57.3	65
19	43.58	41.16	2.42	2.7	60.0	68
23	44.00	42.20	1.80	2.0	61.9	70
29^#)	44.30	43.38	0.92	1.0	63.0	71
29^#)	47.75	48.50	0.00	0.0	63.0	71
29^#)	49.55	50.15	0.00	0.0	63.0	71
^*): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.2 mg CO2/2L. ^#): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

Table 3 CO₂ Production and Percentage Biodegradation of the Reference Item

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^#)(%)
	Blank (mean)	Procedure control
2	46.16	32.38	13.78	15.2	15.2	18
5	43.62	21.14	22.48	24.7	39.9	46
8	41.83	29.67	12.16	13.4	53.3	62
12	42.26	33.73	8.53	9.4	62.6	73
15^#)	43.56	34.93	8.63	9.5	72.1	84
^*): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 86.3 mg CO2/2L. ^#): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Table 4 CO₂ Production and Percentage Biodegradation of the Toxicity Control

Day	HCl(0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^*)(%)
	Blank (mean)	Toxicity control
2	46.16	27.14	19.02	20.9	20.9	12
5	43.62	8.19	35.43	39.0	59.9	34
8	41.83	17.19	24.64	27.1	87.0	50
12	42.26	24.18	18.08	19.9	106.9	61
15^#)	43.56	29.04	14.52	16.0	122.8	70
^*): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and reference item: 174.4 mg CO2/2L (ThCO2test item: 88.1 mg CO2/2L + ThCO2sodium acetate: 86.3 mg CO2/2L). ^#): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'overall remarks'

Interpretation of results:

readily biodegradable

Conclusions:

In a biodegradability test (Modified Sturm Test), performed according to OECD 301B and GLP principles, Cocoa Shell Extract (Pot) was readily biodegradable (73 and 71% biodegradation in duplicate bottles tested).