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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

TEST ORGANISM:
- Origin: mixed population of aquatic microorganisms, origin from a waste water treatment plant, predominantly domestic sewage (Wupper area water authority, STP Odenthal, Germany)
- Concentration of inoculum: 30 mg/L suspendid solids
- Date of collection: 2017-07-24

PRETREATMENT OF THE INOCULUM:
− Before use, the inoculum was stored for three days at room temperature under continuous stirring with aeration
− 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

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/REFERENCE COMPOUND:
- 25 mg of the test item or 25 mg of the reference compound were weighed out on aluminium foil added to the test flasks, filled with 200 mL of mineral medium
- Afterwards the flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item or reference compound/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)

Determinations of test item and inoculum blank were carried out in triplicate and of reference compound in duplicate.

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.9 %, Batch-no.: A0357641

Key result

Parameter:

% degradation (O2 consumption)

Value:

42

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

37

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

26

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

7

Sampling time:

7 d

Parameter:

COD

Value:

2.105 mg O2/g test mat.

Results with reference substance:

Kinetic of reference substance (% degradation):
83 after 7 days
91 after 14 days
94 after 21 days
95 after 28 days

Validity criteria fulfilled:

yes

Remarks:

see: Overall remarks

Interpretation of results:

not readily biodegradable

Conclusions:

Within the test period of 28 days, a degradation of 42 % was determined. Therefore the substance is considered to be “Not Readily Biodegradable“. The reference compound sodium benzoate showed 91 % degradation after 14 days (Neuhahn, 2018).

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 suspension of 100 mg/L test item in a mineral medium, equalling to 50-100 mg ThOD or COD/Litre as the nominal sole source of organic carbon, was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 28 days 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 chemical oxygen demand (COD). 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, one replicate) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms. Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and end of biodegradation and the slope of the biodegradation curve. Determinations of test item and inoculum blank were carried out in triplicate and of 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. Within the test period of 28 days, a degradation of 42 % was determined. Therefore the substance is considered to be “Not Readily Biodegradable“. The reference compound sodium benzoate showed 91 % degradation after 14 days.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge (domestic and industrial)

Details on inoculum:

I. 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) and acctivated sludge from the aeration tanks of a wastewater treatment plant treating predominantly wastewater of industrial origin (WWTP Leverkusen Bürrig)
- Date of collection: 2017-09-18
- Concentration of inoculum (dry weight): 100 mg/L suspended solids (ss) for all treatments

II. Pre-treatment of the inoculum:
− Before use, the inoculum was stored for one day at room temperature under continuous stirring with aeration
− The three sludge types were mixed taking 2 parts from each of the two domestic WWTPS plus 1 part from the industrial WWTP
− 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

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 weighed out on a piece of plastic and both 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

PRE-TREATMENT OF THE REFERENCE COMPOUND:
- 25 mg of the reference compound were weighed out on aluminium foil and both 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 100 mg reference compound/L and a final concentration of suspended solids of 30 mg/L

PRE-TREATMENT OF THE TOXICITY CONTROL:
- 7.5 mg of the test item were weighed out on a piece of plastic and 7.5 mg of the reference compound were weighed out on aluminium foil added to the test flasks, filled with 200 mL of mineral medium
- Afterwards the flask volume was made up to 250 mL with mineral medium plus inoculum to give a final concentration of 30 mg test item and reference compound/L and a final concentration of suspended solids of 100 mg/L

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: 99.9 %; Batch-No.: A0357641)

Key result

Parameter:

% degradation (O2 consumption)

Value:

57

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

52

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

45

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

29

Sampling time:

7 d

Parameter:

COD

Value:

2.105 other: mg O2/mg

Results with reference substance:

The reference compound sodium benzoate showed 80 % degradation after 7 days and 79 % 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 57 % 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 80 % degradation after 7 days and 79 % degradation after 14 days (Spoo-Kloeppel, 2018).

Executive summary:

A study was performed to assess the inherent biodegradability in accordance with the OECD Guideline 302C “Inherent Biodegradability: Modified MITI Test (II)” (1981). A suspension of the substance 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. Within 28 days a degradation of 57 % 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 80 % degradation after 7 days and 79 % degradation after 14 days. All validity criteria of the test method were met. The percent degradation of the reference compound sodium benzoate reached the level of ≥ 40 % after 7 days and ≥ 65 % after 14 days. No toxicity of the test item was observed in the toxicity control.

Description of key information

In a study to assess the ready biodegradation a degradation of 42 % was determined within 28 days. Therefore, the substance is considered to be “Not Readily Biodegradable“ (Neuhan, 2018). In another test according to the OECD Guideline 302 D the substance showed a degradation of 57 %. Therefore the test item is considered to be "Not Inherently biodegradable" (Spoo-Kloeppel, 2018). In both tests the reference compound sodium benzoate showed approx. 80 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed