Salts of 9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthenium and 4,4'-bis[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)diazo] biphenyl- 2,2'-disulfonic acid and 5-amino-4-hydroxy-6-[(4-nitrophenyl)diazo]-3-(phenyldiazo)-3,4-dihydronaphthalene-2,7-disulfonic acid (5:3:1)

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:

2008-08-26 to 2009-04-15 (3rd draft report)

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1

IUPAC Name:

Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1

Constituent 2

Reference substance name:

Acid Yellow 42: 6375-55-9

IUPAC Name:

Acid Yellow 42: 6375-55-9

Constituent 3

Reference substance name:

Acid Black 2: reaction product of 8005-03-6 and 68510-98-5

IUPAC Name:

Acid Black 2: reaction product of 8005-03-6 and 68510-98-5

Constituent 4

Reference substance name:

Basic Violet 1: 8004-87-3

IUPAC Name:

Basic Violet 1: 8004-87-3

Details on test material:

- Name of test material: Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1
- Lot/batch No.: 2008041541
- Expiration date of the lot/batch: 2010-01-31
- Stability under test conditions: see expiry date
- Storage condition of test material: store cool and dry
- Analytical purity: 100 % (96.8 %; UVCB substance)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge, microorganisms from a
domestic waste water treatment plant.
- Laboratory culture:
- Method of cultivation:
- Storage conditions:
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge: 4 g/L
- Initial cell/biomass concentration:
- Water filtered: yes
- Type and size of filter used, if any: cotton wool

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

3 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: Reconstituted water: In deionised water analytical grade salts were added:
a) 4.25 g KH2PO4, 10.875 g K2HPO4, 33.59 g Na2HPO4 x 12H2O, 0.25 g NH4Cl filled up with deionised water to 500 mL volume#
b) 11.25 g MgSO4 x 7H2O filled up with deionised water to 500 mL volume
c) 18.20 g CaCl2 x 2H2O filled up with deionised water to 500 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 1000 mL volume (stock solution was applied on the day of preparation)
- Test temperature: 19.6 - 20.9 °C
- pH: 7.35
- pH adjusted: no
- Aeration of dilution water: 8.8 mg O2/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD flasks
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment: large glass tanks with valve, large glass bottles with valve, narrow necked, 300 ml bod bottles with glass stoppers, funnels and coarse filter papers, moisture content analyzer, centrifuge, balance, self stirring o2 electrode, oxygen and ph meter, aeration system, incubator with thermometer

- Test performed in closed vessels due to significant volatility of test substance:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum was added to 6.80 litres of aqueous test medium.
- Toxicity control: 20.8 mg of F 46 Black and reference item stock solution* (69.0 mL) were mixed into 6.90 litres of aqueous test medium corresponding to 3 mg/L test item (COD of 5.76 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).
* the concentration of the stock solution was 360 mg/L

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

3.6 mg/L

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

0.9

Sampling time:

28 d

Remarks on result:

other: mean value, based on the measured COD

BOD5 / COD results

BOD5 / COD

Parameter:

COD

Value:

7.55 mg O2/g test mat.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 87.5 % after 14 days, and to a mean of 96.7 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the test conditions the percentage biodegradation of F 46 Black reached a mean of 0.9 % after 28 days based on the measured COD of the test item. The test item was not ready biodegradable (level for ready biodegradability is removal of 60 % COD).

Executive summary:

The test item was considered not ready biodegradable.