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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March 2011 - 1 June 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
: suspended solids of the sludge was not within the required range (3-5 g/l) in the first test, therefore sludge was allowed to settle and the liquid was decanted, amount of mineral medium was 8.5 ml/l instead of 10 ml/l; a logical correction was made
equivalent or similar to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
equivalent or similar to guideline
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Constituent 2
Chemical structure
Reference substance name:
Bis[2-(2-ethoxyethoxy)ethyl] cyclohexane-1,4-dicarboxylate
EC Number:
Cas Number:
Molecular formula:
Bis[2-(2-ethoxyethoxy)ethyl] cyclohexane-1,4-dicarboxylate
Details on test material:
- Name of test material (as cited in study report): Neosolue-Aqulio
- Description/Physical state: Clear colourless to very pale yellow liquid
- Molecular formula: C20H36O8
- Molecular weight: 404.5
- Analytical purity: 99.5% (GC)
- Lot/batch No.: TVI-714
- Expiration date of the lot/batch: 07 October 2012 (Retest date)
- Storage condition of test material: At room temperature in the dark

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Method of cultivation:
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (74 and 36 minutes, in the first and second test, respectively) and the liquid was decanted for use as inoculum at the amount of 8.5¹ and 10 ml/l of mineral medium, in the first and second test, respectively.
- Pretreatment: none
- Concentration of suspended solid in the concentrated sludge: 5.6 and 4.7 g/l, respectively (1st and 2nd test)

¹ In the first test the concentration of suspended solids of the sludge as obtained from the municipal sewage treatment plant was not within the range as described by the protocol (3-5 g/l). The original concentration of suspended solids was 5.6 g/l. Therefore, the sludge was allowed to settle and the liquid was decanted for use as inoculum at the amount of 8.5 ml/l instead of 10 ml/l of mineral medium. A logical correction on the amount of inoculum was made. The deviating amount of suspended solids was considered to have no effect on the outcome of this study.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
17 - 20 mg/L
Based on:
test mat.
Initial conc.:
10 - 12 other: mg TOC/l
Based on:
other: organic content was based on the molecular formula (C20H36O8)
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: according to OECD 301
- Additional substrate: no
- Test temperature: between 20.7 and 22.4°C
- pH: 7.6 at start, 7.5 -8.7 at the end
- pH adjusted: yes (in test 1), no (in test 2)
- Aeration of dilution water: yes
- Suspended solids concentration: not reported but expected to meet the guideline criteria because inoculum was prepared in agreement with the guideline
- Continuous darkness: yes (brown coloured bottles)

- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirring, continuous aeration with CO2 free air
- Measuring equipment: none
- Details of trap for CO2 and volatile organics if used: 3 traps, 0.0125 M Ba(OH)2

- Sampling frequency CO2 traps: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension.
- Sample storage before analysis: none

- Inoculum blank: Yes, duplicate
- Reference: Yes, single
- Toxicity control: Yes, single
- Abiotic sterile control: no

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
Two tests were performed:
In the first test the relative biodegradation values calculated from the measurements performed during the test period revealed 31 and 23% biodegradation of Neosolue-Aqulio, for the duplicate bottles tested. Although all criteria for acceptability of the first test were met, the degradation curve of the reference substance (sodium acetate) was not as normally seen at NOTOX. Therefore, there was a doubt on the quality of the sludge and the test was repeated.

The results of the second test were in agreement with the first test and the sodium acetate degradation curve was as expected.

The temperature recorded in a vessel with water in the same room varied between 20.7 and 22.4°C in both tests.

The pH values of the different test media for the first test were in the range 7.6-8.7 and for the second test in the range 7.5 - 7.9.

% Degradationopen allclose all
other: % biodegradation calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 87.2 mg CO2/2l
23 - 31
Sampling time:
29 d
Remarks on result:
other: test 1
other: % biodegradation calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 74.2 mg CO2/2l
9 - 18
Sampling time:
29 d
Remarks on result:
other: test 2
Details on results:
The ThCO2 of Neosolue-Aqulio was calculated to be 2.18 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

The final outcomes of the both tests were in agreement. The criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met in both tests.

The difference of duplicate values for %-degradation of the test substance was always less than 20 (9 at the most).

The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (17 mg CO2/l in the first test and 20 mg CO2/l in the second test).

In the toxicity control more than 25% biodegradation occurred within 14 days (26 and 45%, based on ThCO2, in the first and second test respectively). Therefore, the test substance was assumed not to inhibit microbial activity.

The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC).

BOD5 / COD results

Results with reference substance:
In the positive control (sodium acetate) more than 60% biodegradation occurred within 14 days (61 and 74%, based on ThCO2, in the first and second test respectively).

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
Neosolue-Aqulio was not readily biodegradable under the conditions of the modified Sturm test performed.