Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 2-methoxy-5-nitrophenyl acetate
- Physical state: solid, powder
- Lot/batch No.: 102014
- Storage condition of test material: dry, dark in closed container at the room temperature.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): secondary effluent of the waste water treatment plant of Pardubice processing predominantly municipal sewage.
- Method of cultivation:
- Storage conditions:
- Storage length:
- Preparation of inoculum for exposure: The mineral medium was strongly aerated for 20 minute and let standing for 20 hours at the test temperature.
- Water filtered: yes
- Type and size of filter used, if any: paper filter

Justification of the test system
The inoculum preparation is in conformity with the recommendations of the test guideline.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2.27 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- 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 1000 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 1000 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 1000 mL of water
Solution c)
27.50 g Calcium chloride, anhydrous, (CaCl2) in 1000 mL of water
Solution d)
0.25 g Iron (III) chloride hexahydrate, (FeCl3·6H2O) in 1000 mL of water

- Solubilising agent (type and concentration if used): no
- Test temperature: 20°C +/- 1°C
- pH: 7.4
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 2 x 9 bottles with test substance and inoculated medium, 2 x 9 bottles with reference substance and inoculated medium for the check of inoculum activity, 9 bottles with test substance and inoculated medium for the determination of oxidized nitrogen forms

- Measuring equipment:
- oxygen vessels with glass stopper, volume of approx. 280 mL
- large glass bottles of 5 and 10 L volume for the preparation and inoculation of medium
- closed thermostat
- pH meter WTW pH 539
- analytical balance XS 105 DU (Mettler Toledo)
- equipment for the determination of nitrite
- equipment for the determination of nitrate
- volumetric flasks, flasks, beakers, pipettes and further common laboratory equipment

SAMPLING
- Sampling frequency: At the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of test the appropriate bottles from each series were taken off and they were analysed for dissolved oxygen.

CONTROL AND BLANK SYSTEM
- Inoculum blank: The inoculated mineral medium without test substance was used for the blank determination.

- Toxicity control: The solution was prepared by dosing 90.8 mL of stock solution of test substance and 8.4 mL of stock solution of reference substance into inoculated medium and completed with this medium to the volume of 4 L. The mixture prepared in this way contained 2.27 mg.L-1 of test substance and 2.10 mg.L-1 of sodium benzoate and had COD 6.85 mg.L-1.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (O2 consumption)
Value:
ca. 17.8
Sampling time:
28 d
Details on results:
In 28-day study of ready biological degradability the degradation is 17.8 % of the test substance.
The results of biological degradation are related to calculated COD values of the test and reference substance at the beginning of the test.
Sodium benzoate was used as the reference substance.
COD of the test substance in medium at the beginning of the main test: 3.41 mg.L-1
COD of reference substance in medium at the beginning of the main test: 3.44 mg.L-1
In parallel to the main test the toxicity test was performed.

BOD5 / COD results

BOD5 / CODopen allclose all
Parameter:
COD
Value:
1.497 mg O2/g test mat.
Parameter:
BOD5
Value:
0.21 mg O2/g test mat.
Results with reference substance:
COD of the reference substance: 1.640 mg.mg-1 test material

Any other information on results incl. tables

Quality criteria

Prescribed criteria of the test validity:

The test should satisfy the following quality criteria given in the EU test method:

1. Oxygen depletion in the inoculum blank should not exceed 1.5 mg O2·L-1 after 28 days.

2. The residual concentration of oxygen in the test should not fall below 0.5 mg·L-1 in any time.

3. The difference of extremes of replicate values of the removal of the test substance at the end of the test should be less than 20 %.

4. The percentage degradation of the reference substance should reach the level for ready biodegradability (min. 60 %) by 14 days.

5. If in a toxicity test, containing both the test substance and the reference chemical, less than 25 % degradation should occur in 14 days, the test substance could be assumed to be inhibitory. The test series should be repeated, if possible, using a lower concentration of test substance or a higher concentration of inoculum.

The following values were attained in the current study:

1. Oxygen depletion in the inoculum blank reached 0.51 mg·L-1 during 28th day of the test.

2. The lowest concentration of dissolved oxygen in bottles was 5.16 mg·L-1.

3. The difference of two determinations of the test substance degradation at the end of test was 0.8 %.

4. The percentage degradation of the reference substance has reached the level for the ready biodegradability (min. 60 %) by the 7th day of the test.

5. In the toxicity test, after 14 days of incubation the degradation of the test and reference substance was 42.7 %. Therefore the test substance is not inhibiting for the used inoculum.

All the validity criteria were fulfilled.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The oxidizing properties of test substance, 2-methoxy-5-nitrophenyl acetate, were determined.
The procedure was performed according to the Method A.17 Oxidizing properties (Solids), Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.
Powdered mixtures of test substance and cellulose were prepared in provided weight ratios. Burning time of prepared mixtures was recorded. Burning time was compared with burning time of reference mixture (barium nitrate and cellulose).
The mixture of test substance 2-methoxy-5-nitrophenyl acetate and cellulose showed different behaviour then a mixture of reference substance - barium nitrate Ba(NO3)2 and cellulose.
On the basis of obtained results, it is obvious that the test substance is certainly a better oxidant than the reference substance Ba(NO3)2; thus 2-methoxy-5-nitrophenyl acetate should be considered as a substance with oxidizing properties.
Executive summary:

The test substance, 2-methoxy-5-nitrophenyl acetate, 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 was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out.

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: 3.41 mg·L-1

COD of reference substance in medium at the beginning of the main test: 3.44 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 17.8 % of the test substance, 2-methoxy-5-nitrophenyl acetate, was attained in the end of study.