(3β,20β)-3-acetoxy-11-oxoolean-12-en-29-oic acid

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

17 july 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(3β,20β)-3-acetoxy-11-oxoolean-12-en-29-oic acid

EC Number:

228-475-1

EC Name:

(3β,20β)-3-acetoxy-11-oxoolean-12-en-29-oic acid

Cas Number:

6277-14-1

Molecular formula:

C32H48O5

IUPAC Name:

(2S,4aS,6aR,6aS,6bR,8aR,10S,12aS,14bR)-10-acetyloxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b-dodecahydro-1H-picene-2-carboxylic acid

Test material form:

solid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test System

Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 31 October 2018 (seven days before the main test). The prepared activated sludge was continuously aerated (2L/minute) at the test temperature of 22±2oC, for about 7 days.
Preparation of Activated Sludge Inoculum:
The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium (see Section 5.4) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was: 7.16, just before use: 7.08. A pH adjustment of activated sludge inoculum was not performed.
NaCl (for isotonic saline solution): Manufacturer: lach:ner;
Batch Number: PP/2017/00996,
Expiry date: 24 November 2019
Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2L/minute) activated sludge (in mineral medium ) for 7 days at the test temperature. During the aeration the viability (cell count) of the cultured sludge inoculum was determined by plating 0.1 mL of the 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 dilution of cultures on nutrient agar plates.
The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of ~109 cells/L; therefore, on the day of the test this inoculum was diluted 200000 x with mineral medium to reach the necessary ~104 105cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method.
The inoculum was not pre-adapted to the test chemical.
Nutrient agar: Supplier: MERCK;
Batch Number: VM784250,
Expiry date: 20 April 2022

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

ca. 3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The purpose of this study was to determine the ready biodegradability of the test item Acetylglycyrrhetinic acid. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally, inoculum (containing the filtered inoculum, only) and toxicity (containing both the test item and reference item) controls were examined.
Based on the laboratory’s experience with similar test items, and based on the non-GLP solubility test, the test item was investigated at the concentration of 3.0 mg/L.
The chemical oxygen demand (COD) of Acetylglycyrrhetinic acid/Q (1.70 mg O2/ mg) was measured at the start of the main experiment.
The percentage biodegradation of Acetylglycyrrhetinic acid/Q reached a mean of 15.1 % after 28 days based on its COD. The test item biodegradation value reached its plateau at about the 5th day and from this day only slight changes were observed, within the biological variability range of the applied test system. Under the test conditions the test item biodegradation did not reach the pass level for ready biodegradability that is the removal of 60 % COD in a 10-day window. Based on the obtained values the test item is considered to be not readily biodegradable.
The reference item Sodium benzoate was sufficiently degraded to a mean of 83.7 % after 14 days, and to a mean of 85.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item, a mean of 44.8 % biodegradation was noted within 14 days and 46.5 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

Biodegradation of the Test Item
Under the test conditions the percentage biodegradation of Acetylglycyrrhetinic acid reached a mean of 15.1 % after 28 days based on its measured COD.
In this test the test item biodegradation curve reached its plateau about on the 5th day, from that day only slight increases in biodegradability values occurred. Under the test conditions the test item biodegradation did not reach the pass level for ready biodegradability that is the removal of 60 % COD in a 10-day window. Based on the obtained values the test item is considered to be not readily biodegradable.
The exact nitrogen content of the test item Acetylglycyrrhetinic acid/Q is not known; but supposedly no nitrification occurs during the test and total oxidised nitrogen (nitrate and nitrite) concentration determinations was not performed after the oxygen measurements.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

15.1

Sampling time:

28 d

Any other information on results incl. tables

Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Bottle	Percent of biodegradation after n days of exposure
	[mg/L]	No.	2	5	7	12	14	21	28
Test item		1a	-0.1	13.3	12.0	12.2	14.8	14.4	16.5
	3.0	1b	-0.9	13.1	13.1	13.9	14.4	14.6	13.7
		mean	-0.5	13.2	12.5	13.0	14.6	14.5	15.1
Reference item		2a	54.9	67.6	80.4	79.8	80.3	84.1	83.6
	3.0	2b	54.7	77.4	81.6	85.0	87.1	85.1	87.8
		mean	54.8	72.5	81.0	82.4	83.7	84.6	85.7
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	25.0	40.2	41.6	44.4	44.6	46.7	45.8
		4b	26.5	41.2	42.7	43.8	44.9	44.6	47.2
		mean	25.7	40.7	42.1	44.1	44.8	45.6	46.5

BOD (mg O2/ mg T.i. or R.i.)

Biodegradation % = ----------------------------------------------------------------------------- x100

COD (mg O2/ mg T.i.) or ThOD (NH3) (mg O2/ mg R.i.)

where:

T.i.        = test item

R.i.       = reference item

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item is considered to be not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is 60 % of COD.
In this study total oxidised nitrogen (nitrate and nitrite) concentration determination was not performed after the oxygen measurements.
According to the test guidelines the test item can be assumed as not inhibitory on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.