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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
Study period:
experimental part of study was performed in period from 2013-09-12 to 2013-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was carriedout according to internationaly valid method in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Copper phtalocyanine monosulfonated and disulfonated
EC Number:
700-870-8
Molecular formula:
C32H15N8Cu(HSO3)n where n=1.3-1.6
IUPAC Name:
Copper phtalocyanine monosulfonated and disulfonated
Constituent 2
Reference substance name:
Copper phtalocyanine mono- and disulphonated
IUPAC Name:
Copper phtalocyanine mono- and disulphonated
Constituent 3
Reference substance name:
Hysperse 12
IUPAC Name:
Hysperse 12
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Hysperse 12
- Molecular formula (if other than submission substance): C32H15N8Cu(SO3H)1.4x2.5H2O
- Molecular weight (if other than submission substance): 733.3
- Substance type: multicomponent substance
- Physical state: powder
- Analytical purity: 99.5 % (w/w)
- Impurities (identity and concentrations): water soluble salts0.5 % (w/w)
- Composition of test material, percentage of components:
- Lot/batch No.: 01
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: unlisted
- Storage condition of test material: in closed bottle under the temperature 25 grad. C
- Other:

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
The inoculum contained a mixed population of mocroorganisms obtained from secondary effluent of the STP of Pardubice processing predominantly municipal sewage.
The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using.
The inoculum was in conformity with recomendations of the test guideline.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
2.09 mg/L
Based on:
test mat.
Initial conc.:
3.4 mg/L
Based on:
COD
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The solution of test substance in mineral medium is inoculated with a relatively small number of microorganisms from a mixed population and kept in completely full closed bottles in the dark at constant temperature. 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.

TEST CONDITIONS
- Composition of medium:
Solution A: KH2PO4 8.5 g + K2HPO4 21.75 g + Na2HPO4.2H2O 33.4 g + NH4Cl 0.5 g + water up to 1000 mL
Solution B: MgSO4.7 H2O 22.5 g + water up to 1000 mL
Solution C:CaCl2 27.5 + water up to 1000 mL
Solution D: FeCl3.6H20 0.25 g + water up to 1000 mL
water with conductivity less then 5 microS/cm

Mineral medium: 1 mL A + 1 mL B + 1 mL C + 1 mL D + aerated water up to 1000 mL (1 day stay in laboratory)

Inoculated medium:
The mineral medium was strongly aerated for 20 min. and let standing for 20 h at the test temperature. The volume of waste water for the inoculation of mineral medium was chosen 2.1 mL per 1 L of medium (COD of waste water: 50 mg·L-1).
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: 7.37.

Test substance:
The stock solution of test substance was prepared in concentration 0.1043 g.L-1 of water. From this solution 120 mL (20 mL.L-1) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L.
The solution prepared in this way contained 2.09 mg.L-1 of test substance, and had COD 3.40 mg.L-1. The pH value of solution: 7.43.

Reference substance:
The stock solution of sodium benzoate was prepared in concentration 1.0000 g·L-1of water. From this solution the 12.6 mL (2.1 mL·L-1) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L. The solution prepared in this way contained 2.10 mg·L-1 of sodium benzoate, and had COD 3.45 mg·L-1. The pH value of solution: 7.37.

Toxicity test
The solution was prepared by dosing 80 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 2.09 mg.L-1 of test substance and 2.10 mg.L-1 of sodium benzoate and had COD 6.85 mg.L-1. The pH value of mixture: 7.40.

Control (blank) determination
The inoculated mineral medium without test substance was used for the blank determination.
- Additional substrate:
- Solubilising agent (type and concentration if used): no
- Test temperature: 20 +/- 1.0 °C
- pH: 7.4
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus: large glass bottles of 5 and 10 L for preparation and inoculation medium
- Number of culture flasks/concentration:
2 x 9 bottles with test substance and inoculated medium
2x9 bottles with reference substance and inoculated medium for check of inoculum activity
9 bottles with test substance and inoculated medium for determination of oxidized nitrogens form
2x5 bottles with test and reference substance mixture and inoculated medium for the toxicity test
2x9 bottles with inoculated medium only for the blank determination
- Method used to create aerobic conditions: solutions were prepared from aerated water
- Method used to create anaerobic conditions:
- Measuring equipment: oximeter WTW Oxi 730 with membrane probe CellOx 325
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other: oxidized nitrogen form

STATISTICAL METHODS:
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (O2 consumption)
Value:
7.1
Sampling time:
28 d
Details on results:
tables and graf with study results are attached as background material

BOD5 / COD results

Results with reference substance:
see attached background document

Any other information on results incl. tables

study results - tables and graph - are in attached background document

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: the substance is not fulfilling criteria of ready biodegradability
Executive summary:

The test substance, Hysperse 12, was tested for the ready biological degradability in Closed Bottle Test performed according to: Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008.

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: 3.40 mg·L-1

COD of reference substance in medium at the beginning of the main test: 3.45 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.

Test results

In this 28-day study of ready biological degradability the degradation of 7.1 % of the test substance, Hysperse 12, was attained in the end of study.