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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:
10.11. – 9.12. 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge:
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
Initial conc.:
3.7 mg/L
Based on:
test mat.
Remarks:
(COD 4.67 mg·L-1 )
Initial conc.:
3.63 mg/L
Based on:
test mat.
Remarks:
(COD 4.58 mg·L-1 )
Initial conc.:
3.85 mg/L
Based on:
other: oxidized nitrogen forms
Remarks:
(COD 4.85 mg·L-1 )
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
for nitrification
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
The determination of nitrates and nitrites was carried out in parallel bottles in these time intervals by means of spectrometric methods.
Details on study design:
The volume of waste water for the inoculation of mineral medium was chosen 1.8 mL per 1 L of medium (COD of waste water: 95 mg·L-1). 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 54 mL of modified waste water. The pH value of medium: 7.4.

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

- 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 and it was left to stay one day at laboratory temperature.

Solution a)
Monopotassium dihydrogen orthophosphate, (KH2PO4) 8.50 g
Dipotassium monohydrogen orthophosphate, (K2HPO4) 21.75 g
Disodium monohydrogen orthophosphate dihydrate, (Na2HPO4·2H2O) 33.40 g
Ammonium chloride (NH4Cl) 0.50 g
Water up to the volume of 1 000 mL

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 then 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. Analytical grade reagents were used.

- Test temperature: 20±1.0 °C
- pH: 7.4
- pH adjusted: The right composition of medium was checked by the determination of pH value, which must be 7.4.
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
The bottles were placed into thermostat.
2 x 9 bottles with the test substance and inoculated medium
2 x 9 bottles with the reference substance and inoculated medium for the check of inoculum activity
9 bottles with the test substance and inoculated medium for the determination of oxidized nitrogen forms
2 x 5 bottles with the test and the reference substance mixture and inoculated medium for the toxicity test
2 x 9 bottles with inoculated medium only for the blank determination
- Method used to create aerobic conditions: the solution was prepared from aerated water
- 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: the appropriate bottles from each series were taken off

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, the inoculated mineral medium
- Toxicity control: the mixture contained 3.81 mg·L-1 of the test substance and 2.10 mg·L-1 of sodium benzoate (COD 4.79 mg·L-1, COD 3.44 mg·L-1, initial mixture COD 8.23 mg·L-1 ).






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:
7
Sampling time:
28 d
Key result
Parameter:
COD
Value:
1.26 other: mg·mg-1
Parameter:
ThOD
Value:
1.354 other: mg·mg-1
Remarks on result:
other:
Remarks:
without nitrification
Parameter:
ThOD
Value:
1.771 other: mg·mg-1
Remarks on result:
other:
Remarks:
with total nitrification
Results with reference substance:
Sodium benzoate COD 1.638 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 7.0 % of the test substance, 2-(3´,5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone, was attained in the end of study.
Executive summary:

The test substance, 2-(3´,5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone, 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.

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

The test substance is non-soluble in mineral medium, 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.

COD of the test substance in medium at the beginning of the main test:             3.44 mg·L-1

COD of reference substance in medium at the beginning of the main test:         4.40 – 4.85 mg·L-1

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

Based on 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 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 7.0 % of the test substance, 2-(3´,5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone, was attained in the end of study.

Description of key information

In this 28-day study of ready biological degradability the degradation of 7.0 % of the test substance, 2-(3´,5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone, was attained in the end of study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information