Trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphth[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

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:

2018-03-14 to 2018-04-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphth[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

EC Number:

309-011-8

EC Name:

Trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphth[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

Cas Number:

99688-45-6

Molecular formula:

C45H17Cl3N2O5

IUPAC Name:

1,2,3-trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphtho[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

Test material form:

solid: particulate/powder

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): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Receipt: 2018-03-13
- Preparation of inoculum for exposure: 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 until test start (1 day). The amount of inoculum used to initiate inoculation was 4.5 mL/L (25.0 mg/L dw).
- Initial cell/biomass concentration: approx. 0.52 x 107 CFU/L in the final test solution
- Water filtered: no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

34 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: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: Nominal: 20 - 24, ± 1 °C; Actual: 20.9 – 21.2 °C


- pH:
pH-Value
Start End
Inoculum Control 7.72 1) 7.57
2) 7.55
Functional Control 7.72 7.71
Test Item 7.67 1) 7.55
2) 7.56
Toxicity Control 7.66 7.70

1), 2) = First, second replicate

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: Dark, in an incubator


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined in the incubation vessels by the OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.



SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels



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 concentrations



STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
According to OECD-guideline 301, the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

No preliminary study

Test performance:

Based on the calculated oxygen demand, the test concentration of 34 mg/L, corresponding to an oxygen demand of 50.3 mg O2/L (55.8 mg O2/L with nitrification) in the vessel, was selected.

The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control and functional control) or were prepared directly in brown glass bottles (test item and toxicity control):

• two for the inoculum control (C1, C2)
• one for the functional control (R1)
• two for the test item (P1, P2)
• one for the toxicity control (T1)

Separate replicates of test item and toxicity control will be prepared for measurement of the pH-value at test start:

• one for the test item (PpH)
• one for the toxicity control (TpH)

Inoculum control
The inoculated test medium, consisting of the required volumes of mineral medium stock solutions, ultrapure water and inoculum, was prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.

Functional control
The reference item was weighed out and transferred into a measuring flask, the required volumes of mineral medium stock solutions, ultrapure water and inoculum, were added. 250 mL of this solution were filled in the brown glass bottle of the functional control, using a 250 mL measuring flask.

Test Item
The test item was weighed out for each replicate and directly transferred into the test vessels with 250 mL of the inoculated test medium, using a 250 mL measuring flask.

Toxicity control
The test item was weighed out and directly transferred into the test vessels with 250 mL of the reference item medium (see above), using a 250 mL measuring flask.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with
OxiTop measuring heads and the measuring system was activated.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

11

Sampling time:

28 d

Details on results:

The biodegradation in % was calculated based on the ThOD of 1.48 mg O2/mg test item.

Colony forming units (CFU) of the inoculum were determined prior to test start by standard dilution plate count: 1.16 x 109 CFU/L. The CFU concentration of the inoculum corresponds to approx. 0.52 x 107 CFU/L in the final test solution.

Results of the Functional Control
The pass level for ready biodegradation (≥ 60% degradation) was reached within 3 days. The validity criterion that the degradation should be ≥ 60% after 14 d was fulfilled. Stages of Biodegradation: The adaptation phase changed to degradation phase within 2 days (degradation > 10%), with a maximum of 95% on day 28.

Results of the Toxicity Control
In the toxicity control the biodegradation achieved 45% after 14 days, with a maximum of 46% after 16 days. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Course and Stages of Biodegradation of Functional Control and Test Item
The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. Only the 1st test item replicate reached the 10% level (beginning of biodegradation) within the 28-day-period of the study. Both test item replicates did not reach the 60% pass level until test end. The mean biodegradation on day 28 was 11%.

After a test period of 28 days, the test item is classified as not readily biodegradable within the 28 day period of the study.

BOD5 / COD results

Results with reference substance:

In the toxicity control the biodegradation achieved 45% after 14 days, with a maximum of 46% after 16 days. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Any other information on results incl. tables

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control, Test Item Vat Green 13 and Toxicity Control

		BOD [mg O2/L]
Date	[d]	Inoculum Control	Functional Control		Test Item				Toxicity Control Test Item + Reference Item
			R1		P1		P2		T1
		mean	gross	net	gross	net	gross	net	gross	net
2017-03-15	1	0.9	4.1	3.2	2.3	1.3	2.0	1.1	5.2	4.3
2017-03-16	2	4.5	32.2	27.7	6.9	2.5	6.7	2.2	32.4	27.9
2017-03-17	3	8.4	38.5	30.1	10.4	2.0	10.2	1.8	38.3	29.8
2017-03-18	4	11.7	43.7	32.1	13.5	1.9	12.8	1.2	44.4	32.8
2017-03-19	5	13.8	49.6	35.8	15.7	1.9	15.1	1.3	49.8	36.1
2017-03-20	6	15.7	54.9	39.1	18.0	2.3	17.2	1.5	54.9	39.1
2017-03-21	7	15.8	57.7	41.9	18.7	2.9	18.7	2.9	57.2	41.3
2017-03-22	8	17.1	60.9	43.8	20.1	3.0	20.3	3.2	59.7	42.6
2017-03-23	9	18.1	62.8	44.6	21.2	3.1	21.6	3.5	62.0	43.9
2017-03-24	10	19.1	64.7	45.6	22.4	3.3	22.8	3.8	63.3	44.2
2017-03-25	11	19.8	65.3	45.5	22.5	2.7	24.0	4.2	64.6	44.8
2017-03-26	12	21.3	66.6	45.4	23.9	2.6	25.0	3.7	66.1	44.8
2018-03-27	13	21.8	67.6	45.8	24.5	2.7	25.5	3.7	66.7	44.9
2018-03-28	14	22.1	68.4	46.3	25.6	3.5	26.6	4.5	67.6	45.5
2018-03-29	15	23.0	69.0	46.0	26.8	3.8	26.8	3.8	68.4	45.3
2018-03-30	16	23.3	70.3	47.0	27.7	4.4	27.7	4.4	69.0	45.8
2018-03-31	17	24.7	70.5	45.8	28.6	3.9	28.2	3.5	70.0	45.3
2018-04-01	18	24.8	71.8	47.0	29.6	4.8	28.7	3.9	70.5	45.7
2018-04-02	19	25.7	71.9	46.2	30.4	4.7	29.6	3.9	71.5	45.8
2018-04-03	20	25.9	72.0	46.1	30.9	5.0	29.6	3.7	71.9	46.0
2018-04-04	21	25.5	71.9	46.4	30.9	5.4	29.6	4.1	71.3	45.7
2018-04-05	22	25.8	72.2	46.4	31.0	5.2	29.6	3.8	71.6	45.8
2018-04-06	23	26.1	73.1	47.0	31.4	5.3	29.9	3.8	71.9	45.8
2018-04-07	24	26.6	73.4	46.8	32.4	5.8	30.8	4.1	72.0	45.4
2018-04-08	25	26.9	74.0	47.1	32.5	5.7	31.0	4.1	72.5	45.6
2018-04-09	26	27.0	74.5	47.5	32.9	5.9	31.0	4.0	72.9	45.9
2018-04-10	27	26.8	74.1	47.3	32.7	5.9	31.1	4.3	72.2	45.4
2018-04-11	28	27.0	74.4	47.4	33.5	6.5	31.1	4.1	72.2	45.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

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 Vat Green 13 was determined with non-adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted from 2018-03-14 to 2018-04-11, according to OECD guideline 301 F at the test facility. The test item concentration selected as appropriate was 34 mg/L, corresponding to a ThOD of 50.3 mg O₂/L (55.8 mg O₂/L with nitrification) per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

 

The mean oxygen depletion in the inoculum control was 27.0 mg O₂/L on day 28.

 

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60% was reached within 3 days. The biodegradation reached a maximum of 95% degradation on day 28.

 

In the toxicity control containing both test and reference item, 45% degradation occurred within 14 days, with a maximum of 46% after 16 days. The degradation of the reference item was not inhibited by the test item.

 

The biodegradation of the test item is shown in comparison to the readily biodegradable functional control and the toxicity control. Only the 1^st test item replicate reached the 10% level (beginning of biodegradation) within the 28-day-period of the study. Both test item replicates did not reach the 60% pass level until test end. The mean biodegradation on day 28 was 11%.

 

The validity criteria of the guideline are fulfilled.

The test item is classified as

not readily biodegradable

within the 28 day period of the study.

Biodegradation [%] of the Test Item Vat Green 13 in Comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 34mg/L Test Item	1	6	7	11	13
	2	6	9	8	8
Functional Control 30 mg/L Reference Item		84	92	93	95
Toxicity Control 34mg/L Test Item + 30 mg/L Reference Item		41	45	46	45