[No public or meaningful name is available]

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:

From 2017-08-23 to 2017-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Bis and tris and tetra{N-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino]-N’-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino] methaniminium} [phthalocyanine(bis and tris and tetra)sulfonato-κ4N29,N30,N31,N32]cuprate(II)

Cas Number:

265115-84-2

Molecular formula:

C32H16-τN8Cu(SO3C17H22N3)τ

IUPAC Name:

Bis and tris and tetra{N-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino]-N’-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino] methaniminium} [phthalocyanine(bis and tris and tetra)sulfonato-κ4N29,N30,N31,N32]cuprate(II)

Test material form:

solid: particulate/powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany: it receives predominantly municipal sewage and hardly any industrial chemical waste. Receipt the 2017-08-06.

- Pretreatment: 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 2 ½ 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 (7 days). 10 mL/L of this mixture were used to initiate inoculation.

- Initial cell/biomass concentration: Approx. 1.31 x 10e7 CFU/L in the vessel.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 17 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

ca. 0.612 other: mg C/mg

Based on:

TOC

Initial test substance concentration 3

Initial conc.:

ca. 2.25 other: mg CO2/ mg test item

Based on:

ThCO2

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral salts medium, according to 301B/CO2 Evolution test.
- Test temperature: 22 ± 2°C
- pH: from 7.71 to 7.76 on Day 28.
- Aeration of dilution water: 30 - 100 mL/min.
- Continuous darkness: low light conditions (brown glass bottles)


TEST SYSTEM
- Test vessels: 5000 mL brown glass vessel containing 3000mL of test medium.
- Dispersion treatment: continous stirring
- Application: once at test start
- Number of culture flasks/concentration:
* 2 replicates for the inoculum control
* 1 repicate for the functional control
* 2 replicates for the test item
* 1 replicate for the toxicity control
- Method used to create aerobic conditions: CO2-free air bubbled through the solution at a rate of 30-100 mL/minute.

- Measuring equipment:
pH-Meter, Multi 350i, WTW
Thermohygrograph, LUFFT
Flow meter, Typ DK 800 PV, KROHNE DUISBURG
Medo Compressor, REBIE


- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The produced carbon dioxide is absorbed into the barium hydroxide solution.
Three gas wash bottles, containing 100mL of a 0.0125 mol/L barium hydroxide solution are placed closed to the test vessels. Determination of CO2 carried out by tittration with the barium hydroxide solution at 0.0125 mol/l. For each titration the first gas wash bottle was removed and a new bottle was connected to the last one. 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
- Sampling frequency: D1, D4, D6, D8; D11, D14, D18, D22, D25, D28 and D29.


CONTROL AND BLANK SYSTEM
* 2 replicates for the inoculum control
* 1 repicate for the functional control
* 2 replicates for the test item
* 1 replicate for the toxicity control
- Inoculum control: test medium without test and/or reference item
- Functional control: sodium benzoate at 20 mg/L
- Toxicity control: test item at 17 mg/L and sodium benzoate at 20 mg/L

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

Test performance:

The validity criteria were fulfilled according to the guideline:
•The total CO2 evolution in the inoculum control at the end of the test was < 40 mg CO2/L by day 28 ( 21.7 mg CO2/L on day 28).
•The degradation of the functional control reached the pass level of 60% by day 6 (degradation: 62% on day 6).
•The differences of extremes of replicate values of removal of the test item at the end of the test was less than 20% (1% difference on day 28).
•The degradation of the toxicity control reached the pass level of 25% by day 6 (degradation: 32% on day 6).

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 1

Sampling time:

28 d

Details on results:

Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 1.31x109 CFU/L, corresponding to approx. 1.31x107 CFU/L in the test vessel.

The adaptation phase of the functional control changed within 4 days into the degradation phase (degradation of 10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 6 days and a maximum biodegradation of 88% on day 28. The validity criterion degradation of 60% after 14 days is fulfilled.

Any other information on results incl. tables

Table1: Biodegradation of the Test Item in comparison to the Functional control and the toxicity control

	Biodegradation [%]
	Study Day [d]
	6	14	21	28
Test Item, First Replicate	0	0	0	1
Test Item, Second Replicate	0	0	0	0
Functional Control	62	83	87	88
Toxicity Control test item + reference item	32	44	45	47

 

Table 2:   CO2-Production and Biodegradation after 28 Days

CO2-Production	Functional control	Test Item		Toxicity control test item + reference item
		1	2
Net        [mg/3 L]	113.1	1.2	0.2	114.7
[mg/L]	37.7	0.4	0.1	38.2
Theor.   [mg/3 L]	127.8	114.8		242.6
[mg/L]	42.6	38.3		80.9
Degradation [%]	88	1	0	47

 

Table 3:   CO2-Production and Biodegradation in the Inoculum Control, the Functional Control and the Toxicity Control

Study Day	Date	Inoculum Control	Functional Control			Toxicity Control
		[mg CO2/3 L]	[mg CO2/3 L]		Degr.	[mg CO2/3 L]		Degr.
		mv	Gross	Net Sum	[%]	Gross	Net Sum	[%]
1	2017-08-25	2.5	4.1	1.6	1	4.2	1.7	1
4	2017-08-28	6.3	53.4	48.7	38	50.3	45.7	19
6	2017-08-30	5.4	35.9	79.2	62	37.2	77.5	32
8	2017-09-01	6.5	22.1	94.8	74	19.3	90.3	37
11	2017-09-04	6.2	14.0	102.6	80	15.6	99.7	41
14	2017-09-07	7.1	10.9	106.4	83	13.8	106.4	44
18	2017-09-11	6.6	9.6	109.4	86	8.6	108.4	45
22	2017-09-14	7.7	9.2	110.9	87	9.2	109.9	45
25	2017-09-18	6.7	7.6	111.8	87	7.8	111.0	46
28	2017-09-21	5.2	5.9	112.5	88	5.9	111.7	46
29*	2017-09-22	4.8	5.4	113.1	88	7.8	114.7	47

               Degr. = degradation               mv = mean value           *) results of last two gas wash bottles

 

Table 4:   CO2-Production and Biodegradation in the Inoculum Control and Test Item Samples

Study Day	Date	Inoculum Control	Test Item
			Replicate 1				Replicate 2
		[mg CO2/3 L]	[mg CO2/3 L]			Degr.	[mg CO2/3 L]		Degr.
		mv	Gross		Net Sum	[%]	Gross	Net Sum	[%]
1	2017-08-25	2.5	2.2	0.0		0	2.6	0.1	0
4	2017-08-28	6.3	5.1	0.0		0	4.8	0.1	0
6	2017-08-30	5.4	4.7	0.0		0	4.4	0.1	0
8	2017-09-01	6.5	4.4	0.0		0	3.9	0.1	0
11	2017-09-04	6.2	4.2	0.0		0	4.3	0.1	0
14	2017-09-07	7.1	5.7	0.0		0	5.7	0.1	0
18	2017-09-11	6.6	5.4	0.0		0	5.1	0.1	0
22	2017-09-14	7.7	6.4	0.0		0	6.2	0.1	0
25	2017-09-18	6.7	4.0	0.0		0	6.3	0.1	0
28	2017-09-21	5.2	6.4	1.2		1	3.6	0.1	0
29*	2017-09-22	4.8	4.6	1.2		1	4.9	0.2	0

                  Degr. = degradation         mv = mean value  *) results of last two gas wash bottles

 

Table 5:   pH-Values on Day 28

Inoculum Control		Func-tional Control	Test Item		Toxicity Control
No. 1	No. 2	No.1	No. 1	No. 2	No. 1
7.74	7.75	7.76	7.73	7.72	7.71

 

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 2017-08-23 to 2017-09-22, according to OECD 301 B at the test facility. The test item was tested at a concentration of 17 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 6 days and a maximum biodegradation of 88% on day 28.

 

In the toxicity control containing both test and reference item a biodegradation of 44% was determined within 14 days and it came to 47% after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

 

Both test item replicates did not reach the 10% level (beginning of biodegradation) within the 28-day-period of the study. The mean biodegradation on day 28 was 1%.

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