2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-17 to 2015-04-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Pretreatment/Concentration of sludge:
The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration over night. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. 10 mL/L of this mixture were used to initiate inoculation.
Colony forming units in the test vessel: approx. 10^7 - 10^8 CFU/L

Duration of test (contact time):

28 d

Initial conc.:

28 other: mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: Nominal 22 +/- 2°C (actually measured: 20 - 24 °C)
- Dispersion treatment: Continuous stirring
- Aeration: 30 - 100 mL/min
- Photoperiod: Low light conditions (brown glass bottles)

TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass flasks
- Number of culture flasks/concentration: 1 for the reference item, 1 for toxicity control (test and reference item), 2 for the control, 2 for the test item
- Method used to create aerobic conditions: Aeration with 30 - 100 mL/min
- Measuring equipment: Visual check of aeration twice per day
- Details of trap for CO2 and volatile organics if used:
CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a
0.0125 mol/L Ba(OH)2 solution.
- Course of the study:
The following incubation vessels were prepared:
- two for the test item concentration (P1, P2)
- one for the functional control (R1)
- two for the inoculum control (C1, C2)
- one for the toxicity control (T1)
The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution. Test and reference item were weighed out. The test item was weighed out into small beakers and a defined amount of ultrapure water was added and treated with ultrasound. The test item dispersions and the reference item were transferred to the respective incubation vessels. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air. On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined.


SAMPLING
- Sampling frequency:
Backtitration of the residual Ba(OH)2 with 0.05 N HCL was carried out three times a week during the first ten days and thereafter twice weekly.
- Sampling method:
For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and/or reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentration


STATISTICAL METHODS:
- The theoretical production of carbon dioxide (ThCO2) of the test item and functional control was calculated by the carbon content (1) and the
molecular formula (2), respectively.

ThCO2 [mgCO2/mg] = 3.67 * TOC [mgC/mg test item] (1)

ThCO2 [mgCO2/mg] = (C-Atoms *molecular weight of CO2)/molecular weight of referenz item) (2)


- The produced CO2 was calculated by: 1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2

- The net amount of CO2 produced was calculated by correcting the results of the test item and functional control for endogenous CO2 production
of the inoculum controls.

- The biodegradation was calculated from the ratio theoretical CO2 production to net CO2 production:

Degradation [%] = (net CO2 * 100)/(THCO2 [mg CO2/3L])

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

4

Sampling time:

28 d

Details on results:

Based on the carbon content a ThCO2 of 1.37 mg CO2/mg test item was calculated. A test concentration of 28 mg/L, corresponding to a carbon content of 10.4 mg C/L in the test vessels was selected.

Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior
to test start by standard dilution plate count: approx. 1.20  109 CFU/L, corresponding to
approx. 1.20  107 CFU/L in the test vessel.


In the toxicity control containing both test and reference item a biodegradation of 25 % was determined within 13 days and it came to 34 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in comparison to the readily degradable functional control and the toxicity control. The 10 % level (beginning of biodegradation) was not reached within 28 days. The 60 % pass level was not reached by both test item replicates until test end. The mean biodegradation on day 28 was 4 %.

Under the test conditions the test item is classified as not readily biodegradable within the 28-day period of the study.

In the inoculum control the total CO2 production was 27.4 mg CO2/L after 28 days.

Results with reference substance:

The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation  10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % within 8 days and a biodegradation of 86 % after 28 days. The validity criterion degradation  60 % after 14 days is fulfilled.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the test conditions the test item is classified as not readily biodegradable within the 28-day period of the study.

Executive summary:

The ready biodegradability of the test item Disperse Blue ANT (batch no.: EAG-149-1.0.0) was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2015-03-17 to 2015-04-15 according to OECD 301 B at Dr.U.Noack-Laboratorien. The test item was tested at a concentration of 28 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.4 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature of 20 – 24 °C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO₂ produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO₂ had been purged from the test solutions over a period of 24 hours. The percentage CO₂ production was calculated in relation to the theoretical CO₂ production (ThCO₂) of the test item. The biodegradation was calculated for each titration time.

 

To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 8 days and a biodegradation of 86 % after 28 days.

In the toxicity control containing both test and reference item a biodegradation of 25 % was determined within 13 days and it came to 34 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in comparison to the readily degradable functional control and the toxicity control. The 10 % level (beginning of biodegradation) was not reached by both test item replicates until test end. Both test item replicates did not reach the 60 % pass level until test end. The mean biodegradation on day 28 was 4 %.

 

 

The test item is classified as
not readily biodegradable
within the 28 day period of the study.

 

 

Biodegradation of the Test Item Disperse Blue ANT in Comparison to the Functional Control and the Toxicity Control

	Biodegradation [%]
	Study Day [d]
	6	13	21	28
Test Item, 1^stReplicate	1	1	1	1
Test Item, 2^ndReplicate	0	5	6	6
Functional Control	54	80	82	86
Toxicity Control test item + reference item	12	25	31	34

Reference 2

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 - 27 Jan 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Methods for the Examination of Waters and Associated Materials

Principles of method if other than guideline:

The principle of the biochemical oxygen demand (BOD) test involves measurement of the reduction in dissolved oxygen (DO) in the presence of the test sample and naturally occurring bacteria. The biological "seed" is obtained from a river, downstream of a sewage treatment works treating mainly domestic sewage. A bottle is set up containing dilution water seeded with bacteria (the "blank"). An aliquot of test material or stock solution is added to dilution water to give the required concentration for the test. Initially two different test concentrations are set up.

The DO concentration is measured in all bottles at the start of the test (day 0 of measurement) and 5 days after the start, and the oxygen consumption of the test substance is calculated by comparison of the loss of DO with the loss of DO in the blank. The BOD of the sample is calculated in mg O2 consumed per gram of test substance and degradation is reported as a percentage of the chemical oxygen demand (COD) of the sample.

The COD involves oxidation of the test sample under standard conditions by acidified dichromate, and titration of the residual dichromate with ferrous ammonium sulphate solution.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: obtained from a river, downstream of a sewage treatment works treating mainly domestic sewage

Duration of test (contact time):

ca. 5 d

Initial conc.:

ca. 40 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: deionised water
- Test temperature: average temperature during test period: 18.6°C

Remarks on result:

other: COD/BOD5 measurement

Details on results:

BOD5 of the test solution: 1.1 mg O2/L

Parameter:

BOD5

Value:

30 mg O2/g test mat.

Parameter:

COD

Value:

570 mg O2/g test mat.

Parameter:

BOD5*100/COD

Value:

0.3

Remarks on result:

other: not biodegradable

DO measures [mgO2/L]	Blank	Test
Day 0	8.9	9.1
Day 5	8.6	7.7
loss	0.3	1.4
BOD5 value [mgO2/g]	-	30

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The ratio of BOD5 and COD is 0.3. The substance is not considered to have any potential for biodegradation.

Description of key information

Disperse Rede 167:1 was assessed in a BOD5/COD test and not found to be biodegradable. The structural analogue substance SA02 was used as key study. As almost all disperse dyes, the substance was not found to be readily biodegradable in an OECD 301B test (4% based on CO2 evolution after 28 d). Summarising, Disperse Red 167:1 is not considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater