9-[bis(2-hydroxyethyl)amino]-5-[(3-methylphenyl)amino]-7-phenylbenzo[a]phenazin-7-ium chloride

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

EC Number:

941-946-2

Molecular formula:

C33H31ClN4O2

IUPAC Name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

Details on test material:

- Purity: 89.7 % (w/w) (main component)
- Purity test date: 2014-06-17
- Expiration date of the batch: 2024-06-17

Study design

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 for 23 hours. 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 test substance concentration

Initial conc.:

15 other: mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

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: 20.0 - 23.5 °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 of the incubation vessels. The vessels were
aerated for 24 hours with CO2 free air. After 24 hours, 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. A defined volume of ultrapure water was added, treated
with ultrasound and transferred into the test vessel. This procedure was repeated seven times. The reference item was 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 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

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

13

Sampling time:

28 d

Details on results:

Based on the carbon content a ThCO2 of 2.60 mg CO2/mg test item was calculated. A test concentration of 15 mg/L, corresponding to a carbon content of 10.7 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. 1.04  109 CFU/L, corresponding to
approx. 1.04  107 CFU/L in the test vessel.

The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements. The 28 d-values are shown in comparison to the readily degradable functional control


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 % after 6 days and a biodegradation of 96 % after 28 days. The validity criterion degradation  60 % after 14 days is fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 33 % was determined within 14 days and it came to 35 % 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 1st test item replicate reached 10 % level (beginning of biodegradation) after 21 days. The 2nd test item replicate did not reach the 10 % level until test end. Both test item replicates did not reach the 60 % pass level within 28 days. The mean biodegradation after 28 days was 13 %.

Under the test conditions the test item is classified as not readily biodegradable in the 10-day-window and within 28 days.

In the inoculum control the total CO2 production was 24.4 mg CO2/L after 28 days.

BOD5 / COD results

Results with reference substance:

In the toxicity control containing both test and reference item a biodegradation of 62 % was determined within 13 days and it came to 89 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Applicant's summary and conclusion

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 in the 10-day-window and within 28 days.

Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted according to OECD 301 B.The test item was tested at a concentration of 15 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.7 mg C/L in the test vessels.The test vessels were incubated at low light conditions and at a temperature of 20.0 – 23.5 °C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO₂produced 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 CO₂had been purged from the test solutions over a period of 24 hours. The percentage CO₂production was calculated in relation to the theoretical CO₂production (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 % after 6 days and a biodegradation of 96 % after 28 days.

 

In the toxicity control containing both test and reference item a biodegradation of 33 % was determined within 14 days and it came to 35 % after 28 days. The biodegradation of the reference item was not inhibited by the test itemin 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 1^st test item replicate reached 10 % level (beginning of biodegradation) after 21 days. The 2^nd test item replicate did not reach the 10 % level until test end. Both test item replicates did not reach the 60 % pass level within 28 days. The mean biodegradation after 28 days was 13 %.

 

 

 

The test item is classified as
not readily biodegradable
in the 10-day-window and within 28 days.

 

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

	Biodegradation [%]
	Study Day [d]
	6	14	21	28
Test Item, 1^stReplicate	3	7	14	21
Test Item, 2^ndReplicate	1	1	2	4
Functional Control	66	81	85	96
Toxicity Control Test Item + Reference Item	18	33	34	35