1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

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:

2020-06-04 to 2020-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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

1

Reference substance name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

EC Number:

216-122-4

EC Name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Cas Number:

1502-47-2

Molecular formula:

C6H6N10

IUPAC Name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Test material form:

solid

Details on test material:

Name of test material (as cited in the report): Exolit 775

Specific details on test material used for the study:

Test item Exolit 775
Batch number Ba-Ha-19-025-1
CAS number 1502-47-2
Purity (certified) 95.75% [m/m]
Active ingredient 1,3,4,6,7,9,9b-Heptaazaphenalene-2,5,8-triamine
Sum formula C6H6N10
Molecular weight 218.18 g/mol
Water solubility 0.93 mg/L
TOC* 31.63 %
Appearance white solid
Expiry date 2034-09-06
Recommended storage Store container tightly closed in a cool, well-ventilated place.


* The TOC was calculated at the test facility based on the information provided by the sponsor.

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
- Receipt: 2020-06-02
- 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 with CO2 free air for two days before test start. Further treatment see section ‘preparation of the test vessels’. 3.65 mL/ L of this mixture were used to initiate inoculation (25.0 mg/L dw).

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

33 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 absorption 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 concentration of the test item and the theoretical CO2 production (ThCO2) were calculated based on the carbon content.

The following incubation vessels will be prepared:
- two for the inoculum control (C1, C2)
- one for the functional control (R1)
- two for the test item concentration (P1, P2)
- 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 absorption 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.

The test item and reference item were weighed out. The test item was weighed out into small beakers. A defined amount of ultrapure water was added to the test item. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water. 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:
Back titration 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 reference 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

Key result

Parameter:

% degradation (CO2 evolution)

Value:

11

Sampling time:

28 d

Details on results:

Based on the carbon content a ThCO2 of 1.16 mg CO2/mg test item was calculated. A test concentration of 33 mg/L, corresponding to a carbon content of 10.4 mg C/L in the test vessels was selected.

The adaptation phase of the functional control changed within 4 days into the degradation phase (degradation  10 %). The course of the degradation was fast and the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 92 % on day 28. The validity criterion degradation  60 % after 14 days was fulfilled.

In the toxicity control containing both test item and reference item a biodegradation of 42 % was determined within 14 days, with 49 % 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 in comparison to the readily degradable functional control and the toxicity control. The first replicate reached the 10 % level (beginning of biodegradation) within 14 days. The second replicate did not reach the 10 % level (beginning of biodegradation) within the 28 days study period. Both test item replicates did not reach the 60 % pass level within 28 days. The mean biodegradation on day 28 was 11 %.


In the inoculum control the total CO2 production was 44.3 mg CO2/L after 28 days.

BOD5 / COD results

Results with reference substance:

In the toxicity control containing both test item and reference item a biodegradation of 42 % was determined within 14 days, with 49 % 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:

not readily biodegradable

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 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 2020-06-04 to 2020-07-03 according to OECD 301 B at the test facility. The test item was tested at a concentration of 33 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 22 ± 2 °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 maximum biodegradation of 92 % on day 28.

 

In the toxicity control containing both test and reference item a biodegradation of 42 % was determined within 14 days, with 49 % 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  Figure 1 in comparison to the readily degradable functional control and the toxicity control. The first replicate reached the 10 % level (beginning of biodegradation) within 14 days. The second replicate did not reach the 10 % level (beginning of biodegradation) within the 28 days study period. Both test item replicates did not reach the 60 % pass level within 28 days. The mean biodegradation on day 28 was 11 %.

 

 

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	5	10	12	19
Test Item, 2^ndReplicate	2	2	2	3
Functional Control	59	85	90	92
Toxicity Control Test item + Reference item	31	42	46	49