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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not GLP. According to guideline.
according to
other: AFNOR T 90-312 (1985)
equivalent or similar to
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
limited details on the actual procedure
GLP compliance:
Oxygen conditions:
Inoculum or test system:
other: sewage treatment plant and natural water
Details on inoculum:
- Source of inoculum/activated sludge: sewage treatment plant in Versailles plus natural water from the Seine (France)
- Storage conditions: aerobic
- Storage length: no more than 24 h
- Preparation of inoculum for exposure: the sample from the sewage plant was decanted for 1 h and an aliquote from the surnatant was used the same day. The natural water was used the same day.
- Pretreatment: inoculum was homogenized
Duration of test (contact time):
28 d
Initial conc.:
57.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
- Composition of medium:
Solution a (1 L).
8.5 g KH2PO4
21.75 g K2HPO4
33.4 g Na2HPO4*2(H2O)
2.5 g NH4Cl
Dissolve in H2O, pH 7.2 ± 0.1.

Solution b (1 L).
22.5 g MgSO4*7(H2O)
Dissolve in H2O.

Solution c (1 L).
27.5 g CaCl2 or 36.4 g CaCl2*2(H2O)
Dissolve in H2O.

Solution d (1 L).
0.25 g FeCl3*6(H2O)
Dissolve in H2O. Prepared before use.

Solution e: trace elements (1 L).
39.9 g MnSO4*4(H2O) or 30.23 g MnSO4*H2O
57.2 g H3BO3
42.8 g ZnSO4*7(H2O)
34.7 g (NH4)6Mo7O24 or 36.85 g (NH4)6Mo7O24*4(H2O)
100 mg C10H12FeN2NaO8*3(H2O): obtained by mixing 44.5 mg FeCl3*6(H2O) and 55.5 mg C10H14N2O8Na2*2(H2O).
Dissolve in H2O.

Solution f: yeast extract (100 mL)
15 mg yeast extract
Dissolve in H2O. Prepared before use.

Note. Solution f may be replaced with a solution of growth factors according to SCHLEGEL (100 mL)
Biotin (0.2 mg)
Nicotinic acid (2.0 mg)
Thiamin (1.0 mg)
p-aminobenzoic acid (1.0 mg)
Pyridoxamine (5.0 mg)
Pantothenic acid (1.0 mg)
Folic acid (5.0 mg)
Cyanocobalamin (2.0 mg)
Dissolve in H2O. Prepared before use.

To prepare 1 L medium:
were mixed 10 mL solution a and 1 mL solutions b to f in H2O.
- Solubilising agent: none
- Test temperature: 22 ± 2 °C
- Aeration: yes

- Culturing apparatus:
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: agitation
- Measuring equipment: Dohrmann DC80 carbon analyzer; photooxidation persulfate/UV method
- Test performed in open system: tubes were closed with a gauze/cotton cap

- Sampling frequency: days 0, 4, 7, 14, 21, and 28

- Inoculum blank: yes, 2 vessels
- Abiotic sterile control: yes, 1 vessel
- Toxicity control: yes, 2 vessels
- Inhibition control: yes, 1 vessel

DOC removal percentage (Dt) was calculated according to:
where CB0 and CBt are mean DOC values of the inoculum blank ;
CE0 and CEt are DOC values of sterile control, toxicity control, inhibition control, and test substance
Reference substance:
benzoic acid, sodium salt
37.70 mg DOC/L
% degradation (DOC removal)
Sampling time:
4 d
% degradation (DOC removal)
Sampling time:
7 d
% degradation (DOC removal)
Sampling time:
14 d
% degradation (DOC removal)
Sampling time:
21 d
Key result
% degradation (DOC removal)
Sampling time:
28 d
Results with reference substance:
93-94 % degradation after 4 days

Table 1. Percentage DOC removal

Day MFb Fcl Fc2 Fa Fi Fe1 Fe2 Fe3 MFe
0 1.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
4 1.38 93.58 94.41 2.92 62.29 29.98 31.30 30.36 30.54
7 1.15 94.06 94.20 6.23 86.08 35.90 58.58 33.83 42.77
14 3.78 103.25 99.93 24.43 101.29 104.23 105.02 106.11 105.12
21 0.88 98.83 99.24 15.75 97.96 98.97 97.90 98.78 98.55
28 1.15 99.59 99.59 16.79 98.17 98.87 98.18 98.50 98.51

MFb: average negative control

MFe: test substance average DOC

Fa: abiotic degradation

Fc1, 2: positive control

Fi: inhibition control

Fe 1,2, 3: test substance

Validity criteria fulfilled:
difference among test replica < 20%; TS is not inhibitory
Interpretation of results:
readily biodegradable
Diacetone alcohol (57.5 mg/L) was totally degraded after 14 d in the DOC removal study (28 d).
Executive summary:

Ready biodegradation potential for DAA has been assessed in a non GLP test whose protocol is considered equivalent to OECD 301 A test. In this context, DAA has been demonstrated to be readily biodegradable with 98.51 % biodegradation after 28 days.

Description of key information

In the key study, ready biodegradability was tested according to the guideline AFNOR T 90-312 [equivalent or similar to the DOC Die-Away test (OECD 301 A, EC C.4-A)] in 1988 by Atochem (Boutonnet JC).  The initial test substance concentration was 57.5 mg/L, and the test was performed with a mixture of activated sludge from the wastewater treatment plant at Versailles and water from the river Seine. DOC removal was monitored over a time period of 28 days, and the test substance was completely degraded after 14 days. From the degradation curve, 10% degradation of the test substance was reached after ~1.5 days, and 70% after 10 days, showing that the 10-day window criterion was met. The reference substance, sodium benzoate, achieved 94.4% DOC removal after 4 days and was totally degraded after 14 days.  All conditions for validity were met (differences between test substance duplicates were <20%, the reference substance exceeded 70% degradation within 14 days, and a toxicity control showed that the substance was not inhibitory to the inoculum). The substance is considered to be readily biodegradable under the conditions of the test.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information