Artemisia pallens, ext.

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 13, 2021 to september 16, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

copy of GLP certificate included with date of inspection may 23 and 24, 2019

Specific details on test material used for the study:

Theoretical Oxygen Demand:ThODNH4: 2.710 mg oxygen per mg test item

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The inoculum was a fresh sample of activated sludge and was collected from the aeration tank of a sewage treatment plant receiving predominantly domestic sewage.

Laboratory preparation:
A filtration through a fine sieve (about 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in a mineral medium until sludge was considered free from excess substrate or inhibitor.

Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature.
The sludge was not be pre-adapted to the test item.
Sample site : Water treatment plant "STEP de Lille", 33290 Blanquefort (France); Tank 1
Date of sampling : July 08, 2021
Inoculum physico-chemical characteristic
Suspended solid 4.4 g/L
pH 6.5
Dissolved oxygen 9.1 mg/L
Activated sludge preparation
1: Filtration through a 1 mm sieve
2: 3 Successive decantations into mineral medium
3: Activated sludge aeration during 5 days

Duration of test (contact time):

60 d

Initial conc.:

100.2 mg/L

Based on:

test mat.

Initial conc.:

100.04 mg/L

Based on:

test mat.

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test duration 28 days extended to 60 days
Light intensity Darkness
Temperature 21.7 to 21.9°C,
Culturing apparatus WTW Oxitop
Incubation conditions Closed test flasks incubated in environmental simulation chamber under continuous stirring

test item preparation :
Tthe test item was poorly soluble in water and in order to improve the dissolution, the test item was dissolved in silicone oil AR 20 up to final concentration of 1 % v/v. Around 25 mg of test item was stirred with 2.5 mL silicone oil AR 20 by magnetic stirring.
Then, this solution was directly supplemented or completed into 250 mL with mineral medium and sludge directly in each test vessels.

The test item was tested at 100 mg/L in mineral medium containing 1 % V/V of silicone oil AR 20.

The test medium was the mineral medium, a weakly saline aqueous medium

A measured volume of inoculated medium containing a known concentration of the test item (100 mg/L) as the nominal sole source of Carbon, was stirred in a closed vessel at a constant temperature up to 28 days. At the request of the study monitor, the period was extended to 60 days depending on biodegradation level obtained at T28 with the test item.

Microbial respiration consumes O2 and releases CO2 which were absorbed by soda lime pellets present in the headspace of flasks. This produced a decrease in pressure in the flask proportional to the amount of oxygen taken up by the microbial population during biodegradation, which was expressed as a percentage of ThOD (Theoretical Oxygen Demand).

Allowance was made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without the test item. Furthermore, in order to check the procedure, a reference item control (sodium benzoate 60.2 mg/L + inoculum) and a toxicity control (sodium benzoate + inoculum + test item) were run in parallel.
The pH of the contents was measured at the start and the end of the experiment.


test design
3 flasks: Inoculum Control : Inoculum only
3 flasks: Inoculum silicon Control : Inoculum and silicone oil AR 20 1% v/v
3 flasks: Test item 100 mg/L Test item, silicone oil AR 20 1% v/v and inoculum
2 flasks: Reference item Sodium benzoate and inoculum
2 flasks: Toxicity control Test item, silicon oil AR 20 1% v/v, sodium benzoate and inoculum

The oxygen consumption was automatically recorded for each flask: data were reported in a spreadsheet program. The following calculations were performed:

Biological Oxygen Demand: BOD = (mgO2 uptake by test item - average mg O2 uptake by controls ) / mg test item in vessel

Biodegradation percentage: = (BOD / ThOD ) x 100


The biodegradation of the toxicity control (mixture of the test item and reference item) was calculated with the formula hereafter: (mgO2 uptake by test item and reference item - average mg O2 uptake by controls ) / mg test item and reference item in vessel

Where ThOD of the mixture was obtained as follows:
ThOD = (test it.con. / (test it.conc. + ref.it.conc.) x ThOD test it.) + (ref it.con. / (test it.conc. + ref.it.conc.) x ThOD ref it.)

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

54.53

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

64.33

Sampling time:

60 d

Details on results:

The degradation rate of the test item reached 54.53% (50.71% to 62.15%), following the ThODNH4, after 28 days of incubation. The period was extended to 60 days and the degradation rate of the test item is 64.33% (60.27% to 69.50%).

Results with reference substance:

The reference item “Sodium benzoate” was degraded up to 92.19% (both replicates) after 14 days. Therefore, it confirms the suitability of the used aerobic sludge inoculum.

A biodegradation of 41.60 % and 43.22% after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item) based on ThOD_NH4.

At the end on the test, 60 days of incubation, results were 66.62% and 70.40%.

Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge micro-organisms because degradation was superior to 25% within 14 days (validity criterion).

 

Criteria	Validity for OECD 301F	Validity for the test
Variation between the test item replicates at 60days	< 20%	Yes (Cv = 7.3%)
Biodegradability of the toxicity control within 14 days	> 25%	Yes (41.60% and 43.22%)
Oxygen uptake of the inoculum control in 60 days	< 60 mg/L	Yes (Control: 22.5 to 24.0 mg/L) (Silicone Control: 26.8 to 32.4 mg/L)
Biodegradability of the reference item within 14 days	> 60%	Yes (92.19% both replicates)
pH of the inoculum control at 60 days	6 to 8.5	Yes (Control: 7.3) (Silicone Control: 7.3)

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Based on ThODNH4, the 10-Day window was not reached and the degradation rate of the test item is 54.53% (50.71% to 62.15%), after 28 days of incubation. The period was extended to 60 days and the degradation rate of the test item is 64.33% (60.27% to 69.50%). Under conditions of OECD Guideline 301F, the test item, is not considered to be biodegradable after 28 days of incubation but biodegradable after 60 days.

Executive summary:

The readily biodegradability of the tested substance was assessed according to the OECD Guideline for testing of Chemicals No. 301 F: "Ready biodegradability: Manometric Respirometry Test" over a period of 60 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 60 days of incubation. The test item loading rate was 100 mg/L (corresponding to an oxygen demand of about 271 mg/L (ThODNH4).

The mean biodegradation of the test item at day 28 was 54.53% (ThODNH4), therefore the study was prolonged till day 60. The mean biodegradation at test end after 60 days was 64.33% (ThODNH4). Therefore, test item is considered to be not readily biodegradable based on ThOD NH4 after 28 days but reached degradation rate of 64.33% (ThODNH4) after 60 days.

Description of key information

OECD Guideline 301F, GLP, key study, validity 1:

54.53% after 28 days, 64.33% after 60 days.

The substance is not readily biodegradable but significant mineralisation occurred thus demonstating complete primary biodegradation, and based on the enhanced screening test, extended up to 60 days, the substance is not persistent in the environment.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

One key valid experimental study is available to assess the biodegradation in water of the test substance:

The readily biodegradability of the tested substance was assessed according to the OECD Guideline for testing of Chemicals No. 301 F: "Ready biodegradability: Manometric Respirometry Test" over a period of 60 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 60 days of incubation. The test item loading rate was 100 mg/L (corresponding to an oxygen demand of about 271 mg/L (ThODNH4).

The mean biodegradation of the test item at day 28 was 54.53% (ThODNH4), therefore the study was prolonged till day 60. The mean biodegradation at test end after 60 days was 64.33% (ThODNH4). Therefore, test item is considered to be not readily biodegradable based on ThOD NH4 after 28 days but reached degradation rate of 64.33% (ThODNH4) after 60 days.