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Environmental fate & pathways

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:
25 Oct 2016 to 23 Nov 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
according to guideline
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
GLP compliance:
yes (incl. QA statement)
Akzo Nobel Environmental Chemistry, Zutphenseweg 10, 7418 AJ Deventer, The Netherlands
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (19-10-2016) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L. The inoculum was not pre-exposed to the test substance.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Test procedure: The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing silica gel and inoculum, 10 bottles containing inoculum, silica gel, and test substance, and 6 bottles containing sodium acetate and inoculum. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.

- Nutrients, and stocks: The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw and the content was mixed vigorously. Subsequently 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.
- pH: The pH of the media was 7.3 at the start of the test. The pH of the media at day 28 was 7.2 (control) and 7.3 (control with silica gel and test). The pH was measured using a Eutech Cyberscan pH11 pH meter.
- Test temperature: Temperatures were within the prescribed temperature range of 22 to 24°C. The temperature was measured and recorded with a sensor connected to a data logger.

The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

The following solutions were run in parallel :
- Mineral nutrient solution without test material but with inoculum and silica gel.
- Mineral nutrient solution with only inoculum
- Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.
Reference substance:
acetic acid, sodium salt
Test performance:
The validity of the test is demonstrated by an endogenous respiration of 1.0 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the test substance is 2.9 g O2/g test substance. The sum of isomers with a molecular formula of C9H14O is 99%. The ThOD of sodium acetate is 0.8 mg/mg.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of test substance (multi-constituent) to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.
- Biodegradability: The test substance (multi-constituent) was not biodegraded in the Closed Bottle test and should therefore be classified as not readily biodegradable. Lack of biodegradation does not mean that the test substance (multi-constituent) is recalcitrant in nature. The stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Results with reference substance:
80% degradation of sodium acetate on day 14.

Table: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD)



Oxygen consumption



















Validity criteria fulfilled:
Oxygen concentration was maintained and variability within the test was within the set criteria
Interpretation of results:
not readily biodegradable
Under the conditions test, the substance is not readily biodegradable.
Executive summary:

In order to assess the biotic degradation of Floralozone (multi-constituent), a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

Floralozone (multi-constituent) was not biodegraded in the Closed Bottle test and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that Floralozone (multi-constituent) is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 80% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

The ready biodegradability of Floralozone was investigated in a study conducted in accordance with OECD TG 301D (Closed Bottle test) and GLP (Akzo, 2016). In this study, Floralozone at a test concentration of 2 mg/L was inoculated with activated sludge at a concentration of 2 mg/L. The test substance biodegrades for 2 % during 28 days inoculation. No toxicity control was performed. However, no inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was detected. Therefore, no inhibition of the biodegradation due to the 'high' initial test substance concentration is expected.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The findings in the Akzo (2016) study are confirmed in another biodegradation screening study which was performed in accordance with OECD TG 301C (MITI) and GLP (IFF, 2014). In this study a Floralozone concentration of 100 mg/L was inoculated with an activated sludge concentration of 30 mg/L (suspended solids). The test substance biodegradation was regarded as 0% during the 28 days exposure period. All amount of the test item was converted into Floralozone-alcohol (ethyl-a,a-dimethyl benzeneethanol (p-form and o-form)) and Floralozone-acid (ethyl-a,a-dimethyl benzenepropanoic acid (p-form and o-form)) and in addition seven to nine unknown converted products. None of the converted products were biodegraded under the conditions of the test. In this study also no toxicity control was performed.

 In addition to the studies presented above a ready and inherent biodegradation study are available performed according to OECD 301F and 302C respectively, which are found on the RIFM Database. In these studies no biodegradation was seen. In another BOD test 13.8% biodegradation was seen.