Registration Dossier

Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

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 November 10, 2016 to December 08, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
The inoculum: secondary effluent, microorganisms from a domestic waste water treatment plant.
Origin: The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The secondary effluent used for this study was allowed to settle for an hour, then the decanted effluent was aerated until use (not later than an hour).

The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2 °C according to guideline. The test flasks were placed into an incubator and kept at 21.0 - 22.8 °C, in the dark. The temperature was measured on weekdays during the experiment. The oxygen concentration of test water was 8.9 mg/L at the start of the test. The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.26. The test conditions were measured with suitable instruments and documented in the raw data.
Duration of test (contact time):
28 d
Initial conc.:
3.4 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
PREPARATION OF THE TEST SOLUTIONS
The respective amount of the test substance was weighed in directly to reach the required test substance concentration of 3.4 mg/L.The chosen test substance concentration was based on theoretical oxygen demand (ThODNH4) of 1.78 mg O2/mg test substance (calculated according to equation given in the guidelines) and on the performed 14-d preliminary test.

The components were applied in the amounts/volumes in the test flasks:

1.) Test substance (flasks 1a and 1b), 10 bottles
11.6 mg of test substance was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 3.4 mg/L test substance, with a ThODNH4 of about 6.052 mg O2/L).

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b), 10 bottles
Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution corresponding to 12.240 mg of sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference substance, respectively a ThODNH4 of about 6.012 mg O2/L).

3.) Inoculum Control (flasks 3a and 3b), 10 bottles
Only filtered inoculum was added to 3.40 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b), 10 bottles
The test substance (11.6 mg) and the reference substance stock solution (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 3.4 mg/L test substance (ThODNH4 of 6.052 mg O2/L) and 3.6 mg/L reference substance (ThODNH4 of 6.012 mg O2/L).

Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.


Measurement of Oxygen
The incubation period of the closed bottle test was 28 days. The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Measurement of Temperature
Temperature was measured continuously and registered on weekdays.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
8.3
Sampling time:
28 d
Details on results:
In the toxicity control containing both, the test substance and the reference substance sodium benzoate, a mean of 34.8 % biodegradation was noted within 14 days and 44.3 % biodegradation after 28 days of incubation.
Results with reference substance:
The reference substance sodium benzoate was sufficiently degraded to a mean of 65.8 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

CALCULATION OF RESULTS

- Calculation of BOD

The BOD (mg O2 per mg test substance) expected after each period was calculated as follows:

(mg O2/L of T.i. and/or R.i. – mg O2/L of i.control) / (mg T.i. and/or R.i./L in flask)

where:

T.i. - test substance

R.i. - reference substance,

i.control - inoculum control

- Calculation of Biodegradation %

The percentage biodegradation of the test substance and of the reference substance was calculated as follows:

[BOD (mg O2/mg T.i. or R.i.) x 100] / [ThOD (mg O2/mg T.i) or ThOD (mg O2/mg R.i.)]

where:

T.i - test substance

R.i. - reference substance,

i.control - inoculum control

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
Under the study conditions, the percentage biodegradation of the test substance reached a mean of 8.3 % after 28 days based on the ThOD of the test substance. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test substance is considered not readily biodegradable. According to the test guidelines the test substance can be assumed to be not inhibitory at the applied concentration level of on the activated sludge microorganisms because degradation was >25 % within 14 days.
Executive summary:

A study was conducted to determine the test substance ready biodegradability in a Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4-E and EPA OPPTS 835.3110, in compliance with GLP. The test substance at 3.4 mg/L was inoculated for 28 days in a pre-aerated microbial system collected from a domestic sewage plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. Sodium benzoate (at 3.6 mg/L) was tested simultaneously under the same conditions as the test substance, and functioned as a procedure control. Additionally, toxicity and inoculum controls were used. Sodium benzoate was sufficiently degraded to a mean of 65.8 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum. In the toxicity control containing both the test substance and the reference substance sodium benzoate, a mean of 34.8% biodegradation was noted within 14 days and 44.3% biodegradation after 28 days of incubation. Under the study conditions, the percentage biodegradation of the test substance reached a mean of 8.3% after 28 days based on the ThOD of the test substance. According to the test guidelines the pass level for ready biodegradability is removal of 60% ThOD. Therefore the test substance is considered not readily biodegradable. According to the test guidelines the test substance can be assumed to be not inhibitory at the applied concentration level of on the activated sludge microorganisms because degradation was >25 % within 14 days (Sipos, 2017).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

A study was conducted to determine the test substance ready biodegradability in a Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4-E and EPA OPPTS 835.3110, in compliance with GLP. The test substance at 3.4 mg/L was inoculated for 28 days in a pre-aerated microbial system collected from a domestic sewage plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. Sodium benzoate (at 3.6 mg/L) was tested simultaneously under the same conditions as the test substance, and functioned as a procedure control. Additionally, toxicity and inoculum controls were used. Sodium benzoate was sufficiently degraded to a mean of 65.8 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum. In the toxicity control containing both the test substance and the reference substance sodium benzoate, a mean of 34.8% biodegradation was noted within 14 days and 44.3% biodegradation after 28 days of incubation. Under the study conditions, the percentage biodegradation of the test substance reached a mean of 8.3% after 28 days based on the ThOD of the test substance. According to the test guidelines the pass level for ready biodegradability is removal of 60% ThOD. Therefore the test substance is considered not readily biodegradable. According to the test guidelines the test substance can be assumed to be not inhibitory at the applied concentration level of on the activated sludge microorganisms because degradation was >25 % within 14 days (Sipos, 2017).