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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 10, 2005 to February 08, 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
equivalent or similar to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Type: Mixed population of aquatic microorganisms (activated sludge)
Origin: Aeration tank of a waste water plant treating predominantly domestic sewage (Wupper Area Water Authority)
Date of collection: January 10, 2005
Pretreatment: None
Concentration of inoculum: 30 mg ss/L
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:
- 25 mg test substance was weighed out on aluminium foil. This substance inclusive of the aluminium foil was added to the test vessels to give a test concentration of 100 mg/L test substance.

- Test volume: 250 mL
- Test apparatus: Voith Sapromat
- Type of system: Closed
- Mixing: One magnetic stirrer per test vessel
- Incubation time: 28 d
- Incubation temperature: 22±2°C

- Control (3 replicates)
- Test substance (3 replicates): 100 mg/L
- Reference substance (3 replicates): 100 mg/L
- Toxicity control (2 replicates): Test substance + reference substance

Oxygen consumption was measured on Days 2, 6, 8, 10, 14, 16, 20, 22, 24, 28

- Nitrate-N/Nitrite-N method: Determination of nitrite nitrogen and nitrate nitrogen and the sum of both by flow analysis (CFA and FIA) and spectrometric detection
- Standard: EN ISO 13395
- Test apparatus: Continuous Flow Analyser SKALAR SAN Plus System
Reference substance:
benzoic acid, sodium salt
(purity = 99%)
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
pH of the test vessel: 7.5-7.6

Degradation of test substance:
0% degradation after 2 d
2% degradation after 6 d
3% degradation after 8 d
2% degradation after 10 d
3% degradation after 14 d
3% degradation after 16 d
4% degradation after 20 d
4% degradation after 22 d
4% degradation after 24 d
4% degradation after 28 d
Results with reference substance:
Degradation of reference substance:

48% degradation after 2 d
77% degradation after 6 d
83% degradation after 8 d
85% degradation after 10 d
89% degradation after 14 d
90% degradation after 16 d
92% degradation after 20 d
93% degradation after 22 d
93% degradation after 24 d
94% degradation after 28 d

- No oxygen consumption by nitrification was observed in the flask with test substance at the end of the test.

- The used concentrations of the test substance did not show toxic effects to bacteria.

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
Under the study conditions, the test substance was considered to be not readily biodegradable based on oxygen consumption within 28 d.
Executive summary:

A study was conducted to assess the ready biodegradability of the test substance according to EU Method C.4-D (which is in most parts equivalent to OECD Guideline 301 F), in compliance with GLP.

Activated sludge was inoculated with 100 mg/L of the test substance (in triplicate) in a closed flask at a constant temperature (22±2°C) for up to 28 d. Degradation was assessed by the determination of Biochemical Oxygen Demand (BOD) on Days 2, 6, 8, 10, 14, 16, 20, 22, 24 and 28. A control group, a reference substance group (100 mg/L benzoic acid, sodium salt) and a toxicity test (100 mg/L) were used for validation purposes. The test substance degraded 4% by Day 28. No toxicity of the test substance was observed. The reference compound showed 89% degradation at Day 14.

Hence, under the study conditions, the test substance was considered to be ‘not readily biodegradable’.

Description of key information

Under the study conditions, the test substance was considered to be not readily biodegradable based on oxygen consumption within 28 d (OECD 301).

Key value for chemical safety assessment

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

Additional information