Hydrocotyle asiatica, ext.

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17 July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

Deviations:

no

Principles of method if other than guideline:

Test System
Activated sludge, microorganisms from a domestic waste water treatment plant.
Species:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 22 January 2014.
Origin:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. 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.
Preparation of Activated Sludge Inoculum:
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 reconstituted water (see above) and then aerated until use (in this study 200 mL inoculum was prepared). After preparation the sludge was filtered through cotton wool. The pH of the activated sludge inoculum after preparation was 6.85. A pH adjustment before use was not performed.
The microbial inoculum was prepared on the day of the test and was not pre-adapted to the test chemical.
The microbial inoculum was continuously aerated (2 L/minute) at the test temperature of until use.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocotyle asiatica, ext.

EC Number:

283-640-5

EC Name:

Hydrocotyle asiatica, ext.

Cas Number:

84696-21-9

Molecular formula:

not applicable

IUPAC Name:

Hydrocotyle asiatica, ext.

Test material form:

solid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The Test Groups
Test Item (flasks 1a and 1b):
Based on the chemical oxygen demand (COD) of the test item, 3.27 mg O2/mg [measured in the main test], at the start of the test a suitable volume (500 mL) of the test item solution was thoroughly mixed into the respective volume (5000 mL) of aqueous test medium corresponding to 3 mg/L test item, respectively a COD of about 9.81 mg O2/L.
Remark: In the study plan about 6.24 mg O2/L was calculated instead of 9.81 mg O2/L, because at the calculation the informative preliminary COD was taken into consideration.
Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThODNH3) of sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), the stock solution (360 mg/L) of sodium benzoate (50 mL) was thoroughly mixed into the respective volume of aqueous test medium (5000 mL), corresponding to 3.6 mg/L reference item concentration, respectively a ThODNH3 of about 3.6 x 1.67 = 6.012 mg O2/L.
More studies run in parallel and the corresponding procedure control was common.
Inoculum Control (flasks 3a and 3b)
Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL).
Microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
More studies run in parallel and the corresponding inoculum control was common.
Toxicity Control (flasks 4a and 4b)]
Test (500 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (5000 mL) corresponding to the test item concentration of 3 mg/L [chosen based on the measured informative COD of the test item and the preliminary experiment] and to 3.6 mg/L concentration of the reference item.
In general: microbial inoculum (2.0 mL per litre) was added to each preparation bottle.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Preparation of the Test Solutions
For the preparation of test item test solutions, at first the suitable amount of CENTELLA ASIATICA SELECTED TRITERPENES was suspended in the respective volume of aqueous test medium (reconstituted water) and a stock solution with a concentration of 30.65 mg/L was prepared using (~30 min.) ultrasonic bath.
During the performance of the test the test solutions were mixed by mechanical stirring to ensure a good dispersion (during the incubation period the test solutions were not stirred). The test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.
The concentration of the stock solution in case of sodium benzoate reference item was 360 mg/L.
The Test Groups
1.) Test Item (flasks 1a and 1b):
Based on the chemical oxygen demand (COD) of the test item, 3.27 mg O2/mg [measured in the main test], at the start of the test a suitable volume (500 mL) of the test item solution was thoroughly mixed into the respective volume (5000 mL) of aqueous test medium corresponding to 3 mg/L test item, respectively a COD of about 9.81 mg O2/L.
Remark: In the study plan about 6.24 mg O2/L was calculated instead of 9.81 mg O2/L, because at the calculation the informative preliminary COD was taken into consideration.
2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThODNH3) of sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), the stock solution (360 mg/L) of sodium benzoate (50 mL) was thoroughly mixed into the respective volume of aqueous test medium (5000 mL), corresponding to 3.6 mg/L reference item concentration, respectively a ThODNH3 of about 3.6 x 1.67 = 6.012 mg O2/L.
More studies run in parallel and the corresponding procedure control was common.
3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL).
Microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
More studies run in parallel and the corresponding inoculum control was common.
4.) Toxicity Control (flasks 4a and 4b)]
Test (500 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (5000 mL) corresponding to the test item concentration of 3 mg/L [chosen based on the measured informative COD of the test item and the preliminary experiment] and to 3.6 mg/L concentration of the reference item.
In general: microbial inoculum (2.0 mL per litre) was added to each preparation bottle.

Preparation of Test Flasks
A sufficient number of Winkler flasks were cleaned with 5 – 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for about 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stopper.
Sulphuric acid: Supplier: CARLO ERBA; Batch Number: V2B680142B, Expiry date: February 2018
Potassium iodide: Supplier: REANAL (lach:ner); Batch Number: PP/2012/09667, Expiry date: 08 Febr. 2015
Iodine: Supplier: VWR (PROLABO); Batch Number: 13B070011, Expiry date: February 2018
6.4.2 The Number of Test Flasks
The number test bottles was the following (according to the measurement days):
 at least 14 (+2 reserve) bottles containing the test item and inoculum
 at least 14 (+2 reserve) bottles containing the sodium benzoate and inoculum
 at least 14 (+2 reserve) bottles containing only inoculum (inoculum control)
 at least 14 (+2 reserve) bottles containing the test item and sodium benzoate (toxicity control)
6.5 Measurements
6.5.1 Oxygen Measurements
The incubation period of the closed bottle test was 28 days.
The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].
Oxygen measurements were performed in duplicate on days 0, 2, 7, 12, 14, 21 and 28.
6.5.2 Temperature Measurements
Temperature was measured at least on weekdays in the controlled environment room (during the 28-day incubation period).
6.5.3 Measurement of Total Oxidized N (Nitrite and Nitrate)
The test item does not contain N and total oxidised Nitrogen (nitrate and nitrite) concentrations were not measured after each oxygen measurement.
(Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5 %), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels.).
Measurement of Chemical Oxygen Demand (COD)
The COD (chemical oxygen demand) of the test item was determined in the test facility using COD Cell Test (MERCK),

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

35.8

Sampling time:

28 d

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item is considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % COD in a 10-day window.
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.
According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.