Humic acids and fulvic acids extracted from leonardite, reaction product with formaldehyde, potassium hydrogen sulfite, sodium hydrogen sulfite and potassium hydroxide

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from August 20, 2007 to October 12, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Humic acids, potassium salts

EC Number:

271-030-1

EC Name:

Humic acids, potassium salts

Cas Number:

68514-28-3

IUPAC Name:

68514-28-3

Details on test material:

- Name of test material (as cited in study report): Humic acids, potassium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 16. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

The inoculum contained a mixed population of microorganisms obtained from the secondary effluent of the wastewater treatment plant of Pardubice processing predominantly municipal sewage.
Preparation of the inoculum
The fresh collected wastewater was filtered through paper filter. COD was determined and wastewater was aerated till using.
Justification of the test system
The inoculum preparation was in conformity with the recommendations of the test guideline.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

3.61 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.
Stock solutions for the preparation of mineral medium:
Solution a)
Monopotassium dihydrogen orthophosphate, (KH2PO4) 8.50g
Dipotassium monohydrogen orthophosphate, (K2HPO4) 21.75g
Disodium monohydrogen orthophosphate dihydrate, (Na2HPO4.2H2O) 33.4g
Ammonium chloride (NH4Cl) 0.50g
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±0.1ºC
- pH: 7.4
- pH adjusted: no
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus:
Series of bottles for the test
2 x 9 bottles with test substance and inoculated medium
9 bottles with test substance and inoculated medium for the determination of oxidized nitrogen forms
2 x 9 bottles with reference substance and inoculated medium for the check of inoculum activity
2 x 5 bottles with test and reference substance solution and inoculated medium for the toxicity test
2 x 9 bottles with inoculated medium only for the blank determination
The bottles were lined up into thermostat.

- Measuring equipment:
Instruments and 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 inoLab Level 2
equipment for the determination of COD
equipment for the determination of dissolved oxygen
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 SYSTE
- The inoculated mineral medium only was used for the blank determination.
- Toxicity control:
The solution was prepared by dosing 72 mL of stock solution of test substance and 8.0 mL of stock solution of reference substance into inoculated medium and completed with this medium to the volume of 4 L. The pH value of solution: 7.4.
The mixture prepared in this way contained 3.61 mg.L-1 of test substance and 2.00 mg.L-1 of sodium benzoate and had COD 6.72 mg.L-1. The pH value of mixture: 7.4.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

1.6

Sampling time:

28 d

Details on results:

In 28-day study of ready biological degradability the degradation is 2 % 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.
The test substance had sufficient solubility in used mineral medium. The dosage from the stock solution was carried out.
Sodium benzoate was used as the reference substance.
COD of the test substance in medium at the beginning of the main test: 3.42 mg.L-1
COD of reference substance in medium at the beginning of the main test: 3.30 mg.L-1
In parallel to the main test the toxicity test was performed.
As the chemical composition of test substance is not exactly known the oxidized nitrogen forms for the incidental correction of nitrification were determined .
The test was performed at temperature of 20±1°C with the pH values of solutions 7-8 at the beginning of the test.

BOD5 / COD results

BOD5 / COD

BOD5 / COD 1

Parameter:

COD

Value:

0.95 g O2/g test mat.

BOD5 / COD 2

Parameter:

BOD5

Value:

0.03 g O2/g test mat.

Results with reference substance:

Reference substance:
COD = 1.648 g O2/g test. material

Any other information on results incl. tables

Quality criteria

Prescribed criteria of the test validity:

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 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 0.27 mg.L^-1 on 28th day of the test.

2.The lowest concentration of dissolved oxygen in bottles was 5.92 mg.L^-1.

3. The difference of two determinations of 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 3rd day of the test.

5.In the toxicity test, after 14 days of incubation the degradation of test and reference substance was 33 %. 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

Interpretation of results:

under test conditions no biodegradation observed

Executive summary:

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

The test was performed according to:

Method C.4E - Closed Bottle Test, Directive 92/69/EEC. Published in OJ L 383A, 1992.

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

The test substance had sufficient solubility in used mineral medium. The dosage from the stock solution was carried out.

Sodium benzoate was used as the reference substance.

COD of the test substance in medium at the beginning of the main test: 3.42 mg.L^-1

COD of reference substance in medium at the beginning of the main test:  3.30 mg.L^-1

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

As the chemical composition of test substance is not exactly known the oxidized nitrogen forms for the incidental correction of nitrification were determined .

The test was performed at temperature of 20±1ºC 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 2 % of the test substance, Humic acids, potassium salts, was attained.