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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.01. – 02.02.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
published in O. J. L 142, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
Adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The inoculum contained a mixed population of microorganisms obtained from the secondary effluent of the waste water treatment plant of Pardubice processing predominantly municipal sewage.

- Preparation of inoculum for exposure: The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using. The inoculum preparation is in conformity with the recommendations of the test guideline.
Duration of test (contact time):
28 d
Based on:
other:
Remarks:
not prepared, direct dosing into bottles, see table in box Any other information ...
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
Inoculated medium
The volume of waste water for the inoculation of mineral medium was chosen 1.8 mL per 1 L of medium. 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 60 mL of modified waste water. The pH value of medium: 7.4.
Test substance
The test substance was weighed directly into bottle and the bottles were completed to the whole volume with inoculated medium. Mixtures prepared in this way contained 2.49 – 3.02 mg·L-1 of the test substance (see Table in box Any other information …)
- Composition of medium:
Preparation of 1 litre of mineral medium: 1 mL of solutions a), b), c) and d) were mixed with approx. 500 mL of water, the mixture was replenished with water up to 1 000 mL. The solution was prepared from aerated water.
Solution a)
Monopotassium dihydrogen orthophosphate, (KH2PO4) 8.50 g
Dipotassium monohydrogen orthophosphate, (K2HPO4) 21.75 g
Disodium monohydrogen orthophosphate dodecahydrate, (Na2HPO4·12H2O) 67.21 g
Ammonium chloride (NH4Cl) 0.50 g
Water up to the volume of 1 000 mL
The right composition of medium was checked by the determination of pH value, which must be 7.4.

Solution b) 22.50 g Magnesium sulphate heptahydrate, (MgSO4·7H2O) in 1 000 mL of water
Solution c) 27.50 g Calcium chloride, anhydrous, (CaCl2) in 1 000 mL of water
Solution d) 0.25 g Iron (III) chloride hexahydrate, (FeCl3·6H2O) in 1 000 mL of water

- Solubilising agent (type and concentration if used): The deionized water with conductivity less than 5 uS·cm-1 was used for the preparation of all solutions. The same batch of water was used for each set of the experiments.
- Test temperature: 20±1.0 °C
- pH: of medium 7.4
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
Test substance series: 2 x 9 bottles with test item and inoculum
Reference substance series: 2 x 9 bottles with reference item and inoculum
Blank series: 2 x 9 bottles with inoculated medium only
Toxicity test series: 2 x 5 bottles with test item, reference item and inoculum for toxicity test
Series for nitrification determination: 1 x 9 bottles with test item and inoculum
The prepared dosing solutions for each series were filled in two parallel bottle replicates (except series for nitrification determination). The bottles were placed into thermostat.

- Method used to create aerobic conditions: the solution was prepared from waste water aerated till using
- Measuring equipment: oximeter WTW OXI 730
- Test performed in open system: no

SAMPLING
- Sampling frequency: at the zero-time and at the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test
- Sampling method: 2 bottles (parallel replicate) from the test substance series, from the reference substance series and from the blank series were taken off and the same determination was performed

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, the inoculated mineral medium
- Toxicity control: The test substance was weighed directly into bottle and the bottles were completed to the whole volume with inoculated solution of sodium benzoate. The mixture prepared in this way contained 2.48 – 3.02 mg·L-1 of the test substance and 2.10 mg·L-1 of sodium benzoate.
Reference substance:
benzoic acid, sodium salt
Test performance:
Degradation is determined by analysis of dissolved oxygen over a 28-day period. The following determinations were done simultaneously under the same conditions:
- check of inoculum activity with the reference substance
- toxicity test of the test substance for used inoculum in mixture of the test and reference substance
- determination of oxygen consumption for nitrification
- determination of blank (inoculated mineral medium)

The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of COD.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Key result
Parameter:
COD
Value:
0.97 other: mg·mg-1
Results with reference substance:
Sodium benzoate COD 1.641 mg·mg-1
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In this 28-day study of ready biological degradability the degradation of 0 % of the test substance, Propatyl nitrate, was attained in the end of study.
Executive summary:

The test substance, Propatyl nitrate, was tested for the ready biological degradability in Closed Bottle Test.

Test performance

The test was performed according to:

-             Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008

-             OECD Test Guideline No. 301 D - Closed Bottle Test. Adopted July 17, 1992

 

The results of biological degradation are related to experimentally determined COD values of the test and reference substance at the beginning of the test.

The test substance is non-soluble in the mineral medium, therefore the direct dosing of test substance into each bottle was implemented.

Sodium benzoate was used as the reference substance. The dosage was carried out from the stock solution.

In parallel to the main test the toxicity test was performed.

Based on the fact, that the test substance contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out.

The test was performed at temperature of 20 ± 1 °C with the pH values of solutions 7 – 8 at the beginning of the test.

 

Validity of the test

The prescribed validity criteria in the test were fulfilled.

The test substance was not inhibiting for the used inoculum.

Since all criteria of acceptability were met, this study is considered to be valid.

 

Test results

In this 28-day study of ready biological degradability the degradation of 0 % of the test substance, Propatyl nitrate, was attained in the end of study.

Description of key information

In this 28-day study of ready biological degradability the degradation of 0 % of the test item, Propatyl nitrate, was attained in the end of study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information