Reaction mass of 6-amino-2-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}nicotinonitrile and 2-amino-6-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}heteromonocyclic nitrile

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 -29 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage plant Darmstadt, Germany.

- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. The sediment was re-suspended in test water and aerated overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

103 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Analytical grade salts were added to deionised water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2
H2O, 0.5 g NH4Cl filled up with deionised water to 1000
mL volume
b) 22.5 g MgSO4 x 7 H2O filled up with deionised water to
1000 mL volume
c) 36.4 g CaCl2 x 2 H2O filled up with deionised water to
1000 mL volume
d) 0.25 g FeCl3 x 6 H2O filled up with deionised water to
1000 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d) after storage and before use, one drop of concentrated HCl per litre was added. 10 mL of stock solution a) and 1 mL of the stock solutions b) to d) were combined and filled up to a final volume of
1000 mL with deionised water. The pH-value was 7.6 and therefore no adjustment was necessary.

- Test temperature: 20-22 °C.
- pH:
7.5 – 7.7 (measured at the start of the test)
7.5–8.5 (measured at the end of the test)
- pH adjusted: no
- Suspended solids concentration: 1.5 g dry material per litre were mixed with test water to obtain a final concentration of 30.7 g/L. This suspension was used for the experiment.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL
The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB Sensomat system, Aqualytic Dortmund. The test flasks were closed gas-tight by a measuring head and were incubated in a climatised room under continuous stirring.
Potassium hydroxide solution (45 %) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum control: containing only inoculum (2 bottles)
Procedure control: containing reference substance and inoculum (1 bottle).
Abiotic control: containing test item (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
- Test performed in open system: No

SAMPLING
- Sampling frequency: Daily, from day 1 to day 28.

CONTROL AND BLANK SYSTEM
- Inoculum control: yes
- Abiotic control: yes
- Toxicity control: yes
- Other: Procedure control

Reference substance:

other: sodium benzoate

Parameter:

% degradation (O2 consumption)

Value:

0

Remarks on result:

other: sie details

Details on results:

The test item FAT 41043/A contains nitrogen, therefore the evaluation of biodegradation has to be based ThODNH4 and ThODNO3.

- ThODNH4:
The occurrence of nitrification was considered but not experimentally confirmed. If no nitrification occurs, the 10-day window was not passed. The mean percentage biodegradation at the end of the 28-day exposure period was -16 % (ThODNH4).

-ThODNO3:
Based on ThODNO3 also no 10-day window was passed. The mean percentage biodegradation at the end of the 28-day exposure period was of -16 % (ThODNO3).

- The negative biodegradation of -16 % after 28 days is assumed to reflect the lack of biodegradation, probably caused by the low water solubility of the test substance.

- The oxygen uptake of the inoculum bank was 40 mg/L in 28 days, thus passing the validity criterion.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 75 % after 14 days and to 70 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The degradation rate of FAT 41043/A did not reach 60 % within the 10-day window and after 28 days of incubation. According to the test guideline, FAT 41043/A is considered not to be readily biodegradable.

Description of key information

The ready biodegradability of FAT 41043/A was investigated according to OECD Guideline 301 F, EU Method C.4-D and GLP principles (NOTOX study 498117). No biodegradation of FAT 41043/A was observed after 28 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

NOTOX study Nr. 498117:

FAT 41043/A was not ready biodegradable under the conditions of the 'Manometric Respirometry Test.

NOTOX study Nr. 497918:

Due to the physical chemical properties of FAT 41043/A, especially the limited solubility in water, performance of a Zahn-Wellens/EMPA test was not possible. NOTOX study Nr. 498116: According to the general principle of the BOD5 test a solution of FAT 41043/A of at least 2 mg/l in mineral medium should be inoculated and incubated under aerobic conditions in the dark or in diffuse light. Due to the physical chemical properties of FAT 41043/A, especially the limited solubility in water, performance of a BOD5 test was not possible. Therefore, it is not possible to perform the BOD5 test (EEC Directive 440/2008, Part C.5. 2008; ISO Standard 5815, 2003).