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Biodegradation in water: screening tests

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biodegradation in water: screening test, other
inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
the activated sludge was not fed during the holding period of maximum seven days, the test was run at 22 °C, the test water composition was slightly changed, biological oxygen demand was monitored, no test item specific analysis was performed
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-SAT 070029
- Physical state: viscous, light yellow to amber colored liquid
- Analytical purity: > 98%
- Impurities (identity and concentrations): 1 - 2 % Nonyl-4-H-1,3-benzodioxi
- Lot/batch No.: JB 620-70-1
- Expiration date of the lot/batch: 11.10.2011
- Storage condition of test material: at about 20°C in the dark
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: obtained from a wastewater treatment plant (ARA Ergolz Il, Füllinsdorf, Switzerland) treating predominantly domestic wastewater
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. During the holding period of three days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge defined amounts were added to test water to obtain a final concentration of 100 mg dry material per liter.
- Concentration of sludge: 4 g solids/L
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:
- Composition of medium: according to OECD guideline 301F
- Test temperature: 22 'C, maintained with a built-in thermostat and checked once per week.
- pH: 7.1 - 7.6
- Continuous darkness: yes
- Other: incubation under continuous stirring in a SAPROMAT D12

- Culturing apparatus: 500 mL Erlenmeyer flasks
- Number of culture flasks/concentration: 2

- Toxicity control: yes (1 replicate)
Reference substance:
benzoic acid, sodium salt
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The biochemical oxygen demand (BOD) of the test substance in the test media was in the normal range found for the inoculum controls. Consequently, the test substance was not biodegradable under the test conditions within 28 days.
Results with reference substance:
In the procedure controls, the reference item (sodium benzoate) was degraded by an average of 79% and 84% by Exposure Day 7 and 14, respectively; thus, confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 86%.

In the toxicity control, containing both the test substance and the reference item sodium benzoate, the test substance had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 30 mg/L

Validity criteria fulfilled:
Interpretation of results:
not inherently biodegradable
In a study according to OECD test guideline 302C under GLP the test substance was not inherently biodegradable.
Executive summary:

The test substance was tested for its biodegradability in an OECD 302C study using activated sludge. The study was performed under GLP. There was no inhibitory effect on the sludge at the test concentration of 30 mg/L. As no biodegradation was observed over 28 days (based on BOD), the test substance was not inherently biodegradable.

Description of key information

Not biodegradable: 0% biodegradation in 28 days test period (OECD 302C)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One test investigating the inherent biodegradability of Benzaldehyde, 2-hydroxy-5-nonyl, oxime, branched (CAS 174333-80-3) is available. The study was performed according to GLP and OECD guideline 302C using non-adapted activated sludge as inoculum (Seyfried 2007). The biochemical oxygen demand (BOD) of the test substance in the test media was in the normal range found for the inoculum controls. Thus, the test substance was not biodegradable under the test conditions within 28 days.

In addition, several screening tests for 4-nonylphenol are available (see table 2). In general, these tests show that 4-nonylphenol is not readily biodegradable according to OECD criteria (MITI 2002, Huels 1996 a,b). Degradation is observed if adapted inoculum was used (Staples et al. 1999, 2001) indicating 4-nonylphenol to be inherently biodegradable. However, the degradation potential is depending on several factors, such as the branching of the alkyl chain (see Corvini et al. 2006).

Table 2: biodegradation test results for 4 -nonylphenol






0% after 14 days


MITI 2002


47.5% after 28 days (adapted inoculum)


Staples et al. 2001


57.4 – 68.4% after 28 days (adapted inoculum)


Staples et al. 1999


0 % after 32 days (with and without emulsifier)

with adopted inoculum (adaption time 7 weeks):

0 % after 40 days (without emulsifier)

78 % after 40 days (with emulsifier)


(EU RAR 2002, Huels 1996a; Huels 1996b)

* Data obtained from the respective dossier published on the ECHA data base and from EU RAR 2002.



MITI 2002. Biodegradation and Bioconcentration of Exisiting Chemical Substances under the Chemical Substances Control Law. Information on the chemical published in the Official Bulletin of Economy, Trade and Industry.

Staples CA, et al. 2001. Ultimate biodegradation of alkylphenol ethoxylate surfactants and their biodegradation intermediates. Environ Toxicol Chem 20(11): 2450–2455

Staples CA, et al. 1999. Measuring the biodegradability of nonylphenol ether carboxylates, octylphenol ether carboxylates, and nonylphenol. Chemosphere 38(9): 2029-2039

EU RAR.2002. European Union Risk Assessment Report 4-nonylphenol (branched) and nonylphenol.

Huels. 1996a. Determination of the biological degradability of nonylphenol in the modified sturm test (EEC Directive 79/831 ENV/283/80) Report ST-3/84

Huels. 1996b. Determination of the biological degradability of nonylphenol in the modified sturm test (EEC Directive 79/831 ENV/283/80) Report ST-3a/84.