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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:
March 20th to April 30th, 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
5-methyl-2-(isopropyl)cyclohexyl nicotinate
EC Number:
EC Name:
5-methyl-2-(isopropyl)cyclohexyl nicotinate
Cas Number:
Molecular formula:
5-methyl-2-(isopropyl)cyclohexyl nicotinate

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Camposampiero (PD) WT from the aerobic aeration re-circulation.
- Inoculum final concentration: suspended solids 27.6 mg/l, microbiological count UFC/l 6.0x10^7
- Microbiological count: colony counting is performed on agar at 36 °C and 22 °C in Petri dishes. The thermostatted agar is poured into each plate and allowed to solidify and the plates are incubated for 44 h (36 °C) and 68 h (22 °C).
- Suspended solids determination: determined by filtration on 0.45 μm pre-weighted filter of 5 ml of inoculum and then drying the pre-weighted filter at 105 °C by constant weight. The results are in mg/l from the ratio between the residue on initial volume.
- Preparation of inoculum for exposure: the inoculum was washed with mineral medium and kept in aeration for 5 days inside the mineral medium.
Duration of test (contact time):
28 d
Details on study design:
- Composition of medium: in a 50 l container 500 ml of a) stock solution were added to 20 l of water, then 50 ml of b) c) d) stock solutions were added; the volume is then adjusted to 50 litre. Stock solution a): In a 1 l flask were weighted and then adjusted to the 1 l volume with water: 8.5 g of potassium dihydrogen orthophosphate (KH2PO4), 21.75 g dipotassium hydrogen orthophosphate (K2HPO4), 33.4 g disodium hydrogen orthophosphate dihydrate (Na2HPO4. 2H2O), 0.5 g ammonium chloride (NH4Cl), pH 7.4. Stock solution b): in one 1 l flask were weighted and then adjusted to the 1 l volume with water: 36.4 g calcium chloride dihydrate (CaCl2. 2H20), Stock solution c): in a 1 l matrass were weighted and then adjusted to the 1 l volume with water: 22.5 g magnesium sulphate eptahydrate (MgSO4.7H20). Stock solution d): in a 1 l matrass were weighted and then adjusted to the 1 l volume with water: 0.25 g iron (III) chloride hexahydrate (FeCl3. 6H20) with water and mixed.
- Test temperature: 20-24 °C
- Sample solutions at the end of the test: pHstart= 7.4 pHend = 7.3 B - pHstart= 7.4 pHend = 7.2
- Aeration of dilution water: the system was aerated in continuous with CO2 free air with a flux between 30-100 ml/min.

- Culturing apparatus: bottles of 5-l volume, equipped with an aeration tube.
- Number of culture flasks/concentration: 1. Bottle 1 e 2 containing test substance.
- Trap and measurement of CO2 and volatile organics if used: every single bottle was connected to 3 Drechsel laid out in line containing 100 ml of BaOH 0.0125 M Τhe CO2 produced is trapped in barium hydroxide. Quantitative determination of CO2 was obtained titrating exceeding Ba(OH)2 with HCl 0.05 M using phenolphthalein as indicator.
- Preparation of the test system: in every bottle 3 litres of mineral medium were loaded with 10 ml of activated sludge and aerated for 24 hr with CO2 free air to reach suspended solids less than 30 mg/l. After 24 hr the test sample and the reference compound were added to yield a final concentration of 10-20 mgTOC/l. Every bottle was adjusted to the final volume of 3 l with mineral medium.

- Sampling frequency: during the first ten days titrations were made every second or third day and then at least every fifth day.

- Inoculum blank: bottles 3 and 4 containing only inoculum
- Reference substance sterile control: bottles 5 and 6 containing the reference substance
- Toxicity control: bottle 7 containing test substance, reference compound and inoculum.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
Biodegradation of toxicity control (14 days) > 25 %
CO2 evolved from the blank is less than 70 mg/l
The difference of the results of replicate values is inferior to 20 % for the Test and Reference Substance.

BOD5 / COD results

Results with reference substance:
Biodegradation (28 days): 87.1 %

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Biodegradation (28 days) = 76.7 %
Executive summary:

The biodegradation of the substance is evaluated according to the OECD Guideline 301B. A measured volume of inoculated mineral medium, containing a known concentration of the test substance as the nominal sole source of organic carbon is aerated by the passage of carbon dioxide-free air at a controlled rate. Degradation was followed over 28 days by determining the carbon dioxide produced. The CO2 is trapped in barium hydroxide and is measured by titration of the residual hydroxide. Toxicity, inocolum and reference control run in parallel. All validity criteria were met.

10 days immediately following the attainment of 10 % biodegradation the biodegradability of the test item is superior to the 60 % (10-d windows).

Biodegradation (28 days) = 76.7 %

The test material presents a 60 % CO2 production and this pass value is reached in a 10-d window within the 28-d period of the test.

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