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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge (domestic and industrial)
Details on inoculum:
Activated sludge from an aeration tank of two different wastewater treatment plants treating predominantly domestic wastewater (Wupper area water authority, WWTP Odenthal and WWTP Cologne-Stammheim).
Activated sludge from the aeration tanks of a wastewater treatment plant treating predominantly wastewater of industrial origin (WWTP Leverkusen Bürrig).
The three sludge types were mixed taking 2 parts from each of the two domestic WWTPS plus 1 part from the industrial WWTP.
Pre-treatment of inoculum:
-The three sludges of two different origins were mixed as described before.
-The combined sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
-An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension with a defined concentration of suspended solids.
-The calculated amount of sludge, needed to achieve 300 mL of this stock suspension of 5 g dw/L, was dissolved in mineral medium and then filled up to a defined end volume.
-Before use, the inoculum was stored for one day at room temperature under continuous shaking with aeration.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Pre-treatment of the test item
7.5 mg of the test item were added to the test flasks, filled with 200 mL of mineral medium. Afterwards the volume was made up to 250 mL with mineral medium plus inoculum to give a final concentration of 30 mg test item/L and a final concentration of suspended solids of 100 mg/L.

A measured volume of inoculated mineral medium, containing a known concentration of 30 mg/L of the test item was stirred in a closed flask at a constant temperature (25 ± 2 °C) under aerobic conditions in the dark.
The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated respirometer flasks. Evolved carbon dioxide was absorbed in sodium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of the theoretical oxygen demand (ThOD).
The test lasted for 28 days.
The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures.
A toxicity control (test item and reference compound mixed) was not run in parallel, because the chosen concentration of the test item was not inhibitory to microorganisms (see study 2012/0061/07).
Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and for the inoculum blank were carried out in triplicate and for the reference compound in duplicate.
The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks.
Reference substance:
benzoic acid, sodium salt
Remarks:
Purity: 99.7 %
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
28 d
Details on results:
6 % degradation after 7 days
7 % degradation after 14 days
6 % degradation after 21 days
7 % degradation after 28 days

pH after 28 days: 7.0 - 8.2
Parameter:
COD
Value:
1.337 other: mg O2/mg test mat.
Results with reference substance:
The per cent degradation of the reference compound sodium benzoate reached the level of ≥ 40 % after 7 days, but remained 2 % below the level of ≥ 65 % after 14 days, caused by high activity of the sludge in the blank control. As the degradation was above 40 % after 7 days (65% degradation on day 7) and the test item showed only 7 % biodegradation after 28 days, the test was regarded to be valid.
Validity criteria fulfilled:
yes
Remarks:
The degradation of the reference compound reached the level of ≥ 40 % after 7 days, but remained 2 % below the level of ≥ 65 % after 14 days.As the test item showed only 7 % biodegradation after 28 days, the test was regarded to be valid.
Interpretation of results:
not inherently biodegradable
Conclusions:
Within 28 days a degradation of 7 % was determined according to the OECD Guideline 302 C. Therefore the substance is considered to be "Not Inherently Biodegradable".
Executive summary:

To test for its inherent biodegradability potential, the substance was incubated for 28 days in continuously stirred 250 ml closed flask (three replicates) in the dark with an inoculum of mixed population of aquatic microorganisms (activated sludge) partially from domestic wastewater treatment plant and partially from industrial origin. In this assay, biodegradation was estimated by biological oxygen demand (BOD) over time. BOD was determined daily measuring the drop in pressure in the automated respirometer flasks. The incubation temperature was 25 ± 2 °C, pH was 7.0 - 8.2. The concentration of inoculum was 100 mg /L and the one of test substance was 30 mg/L. Degradation was calculated by subtracting the amount BOD in the negative (inoculum only) control from that in the test material or positive control at any given time point and divided by the chemical oxygen demand (COD) or theoretical oxygen demand (ThOD). The per cent degradation of the reference compound sodium benzoate reached the level of ≥ 40 % after 7 days, but remained 2 % below the level of ≥ 65 % after 14 days, caused by high activity of the sludge in the blank control. As the degradation was above 40 % after 7 days (65% degradation on day 7) and the test item showed only 7 % biodegradation after 28 days, the test was regarded to be valid. The test item is considered to be "Not Inherently Biodegradable".

Description of key information

Within 28 days a degradation of 7 % was determined in a study for inherent

biodegradability. Therefore the substance is considered to be "Not Inherently Biodegradable". As a conclusion, the substance is considered as not readily biodegradable as well, because there is no indication that ready biodegradability will be observed in a ready biodegradability test after the poor inherent biodegradability ratio of the substance in the present study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information