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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-11 till 2012-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
This test method is in all essential parts identical with OECD Guideline 301 F (adopted July 1992).

GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
TEST ORGANISM:
- mixed population of aquatic microorganisms, origin from a waste water treatment plant, predominantly domestic sewage (Wupper area water authority, STP Odenthal, Germany)
- concentration: 30 mg/L suspendid solids
- date of collection: 2011-11-15

PRETREATMENT OF THE INOCULUM:
− the 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 (activated sludge) of 3 g dw/L
− the calculated amount of sludge, needed to achieve 300 mL of this stock suspension, 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.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
PRETREATMENT OF THE TEST ITEM:
- 25 mg of the test item were weighed out on a piece of plastic
- test item and the piece of plastic were added to the test flasks, which had already been filled with 200 mL of mineral medium
- afterwards the flask content was made up to 250 mL with mineral medium containing inoculum to give a test concentration of 100 mg test item/L.

EXPOSURE CONDITION:
- test volume: 250 ml
- incubation temperature: 22 +/- 1 °C
- mixing: 1 magnetic stirrer per test vessel
- test apparatus: Oxi Top System (WTW)

Reference substance:
benzoic acid, sodium salt
Remarks:
Purity: 99.7 %, Batch-no.: 1438955
Parameter:
% degradation (O2 consumption)
Value:
19
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
36
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
54
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
28 d
Parameter:
COD
Value:
1.935 mg O2/g test mat.
Results with reference substance:
Kinetic of reference substance (% degradation):
80 after 7 days
88 after 14 days
90 after 21 days
91 after 28 days
Validity criteria fulfilled:
yes
Remarks:
see: Overall remarks
Interpretation of results:
other: Not Readily Biodegradable
Conclusions:
Within the test period of 28 days, a degradation of 60 % was determined for the test substance. Therefore, Ester PSA + DEG is considered to be “Not Readily Biodegradable“ (Neuhahn, 2011).
Executive summary:

The study was conducted in accordance with the EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test) which is in all essential parts identical with OECD Guideline 301 F to assess the ready biodegradability. A solution of the test substance in a mineral medium was inoculated and incubated for 28 d under aerobic conditions and pH-values were in between 6.0 and 8.5. During this period, degradation was followed by BOD determinations. The test substance showed 60 % biodegradation after 28 days and therefore has to be classified as "Not Readily Biodegradable".

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-27 till 2012-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
A solution of Ester PSA + DEG in a mineral medium was inoculated with 40 % sludge of each of two different municipal sewage treatment plants (STP) and 20 % sludge of an industrial STP and incubated under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge (domestic and industrial)
Details on inoculum:
- Origin: 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
- Date of collection: 2012-02-27
- Concentration of inoculum (dry weight): 100 mg/L suspended solids (ss) for all treatments

Pre-treatment of the 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:
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 chemical oxygen demand (COD).
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 2011/0121/13).
Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item were carried out in quadruplicate, for inoculum blanks 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.
The degradation rate of the test item was set in relation to its experimentally determined COD. As the COD implicitly covers the oxygen demand for the nitrification process, there was no need to take additional correction measures into account.

Exposure conditions
- Test volume : 250 mL
- Test apparatus : OxiTop System (WTW)
- Mixing : 1 magnetic stirrer per test vessel
- Incubation time : 28 days
- Incubation temperature : 25 ± 2 °C
Reference substance:
benzoic acid, sodium salt
Remarks:
(purity 99.7 %, batch no.: 1438955, 100 mg/L)
Parameter:
% degradation (O2 consumption)
Value:
45
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
68
Sampling time:
28 d
Details on results:
A solution of Ester PSA + DEG in a mineral medium was inoculated with 40 % sludge of each of two different municipal sewage treatment plants (STP) and 20 % sludge of an industrial STP and incubated under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations.
Parameter:
COD
Value:
1.935 other: mg O2/mg
Results with reference substance:
The reference compound sodium benzoate showed 71 % degradation after 7 days and 74 % degradation after 14 days.

After 28 days the pH values of different flasks are range of 7.0 - 8.1.

Validity criteria fulfilled:
yes
Remarks:
The percent degradation of the reference compound sodium benzoate reached the level of ≥ 40 % after 7 days and ≥ 65 % after 14 days.
Interpretation of results:
not inherently biodegradable
Conclusions:
Within 28 days a degradation of 68 % was determined according to the OECD Guideline 302 D. Therefore the test item is considered to be "Not Inherently Biodegradable". The reference compound sodium benzoate showed 71 % degradation after 7 days and 74 % degradation after 14 days (Neuhahn, 2011).
Executive summary:

To test for its inherent biodegradability potential, the substance was incubated for 28 days in continuously stirred 250 mL closed flask (four replicates) in the dark with an inoculum of mixed population of aquatic microorganisms (activated sludge) partially from two domestic wastewater treatment plants and partially from one industrial wastewater treatment plant. 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 - 8.1. The concentration of inoculum was 100 mg/L and the one of test substance was 30 mg/L. Degradation was calculated by substracting 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 the theoretical oxygen demand (ThOD). The 28 -day degradation was 68 % and for the positive control (with reference substance) the biodegradation was 74 % after 14 days. In conclusion the substance is not inherently biodegradable.

Description of key information

Within the test period of 28 days, a degradation of 60 % was determined for the test substance. Therefore, the test item is considered to be “Not Readily Biodegradable“ (Neuhan, 2011).
Within 28 days a degradation of 68 % was determined according to the OECD Guideline 302 D. Therefore the test item is considered to be "Not Inherently Biodegradable" (Neuhahn, 2011).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Ready biodegradation:

The test substance showed only 60 % biodegradation after 28 days and therefore has to be classified as "Not Readily Biodegradable".

Inherent biodegradation:

The test substance showed 68 % degradation according OECD guideline 302 D and therefore to be classified as "Not Inherent Biodegradable".