Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

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:

2016-06-23 to 2017-03-13

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: International Standard ISO 9439

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

2008-05-30

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

EC Number:

915-758-6

Molecular formula:

C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na

IUPAC Name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

Appearance:
- physical state: solid
- color: dark brown

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 27 June 2016 from the aeration tank of the plant. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm.
- Preparation of inoculum for exposure: To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 24 hours at 22 ± 2 °C.
- Pretreatment: At the day of exposure the suspension was washed one time with drinking water. Therefore the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drinking water.
- Concentration of sludge: 6.0 g/L dry weight

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:
Solution A:
KH2PO4: 8.50 g
K2HPO4: 21.75 g
Na2HPO4 x 2 H2O: 33.40 g
NH4Cl: 0.50 g
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.

Solution B:
CaCl2 x 2 H2O: 36.40 g
The compound was dissolved with deionized water to 1000 mL.

Solution C:
MgSO4 x 7 H2O: 22.50 g
The compound was dissolved with deionized water to 1000 mL.

Solution D:
FeCl3 x 6 H2O: 0.25 g
The compound was dissolved with deionized water to 1000 mL.

15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.

- Test temperature: 22 ± 2 °C
- pH: 7.2 - 7.4
- pH adjusted: yes, 7.4 ± 0.2

TEST SYSTEM
- Culturing apparatus: 2 L incubation bottles filled up to a volume of 1.5 L.
- Number of culture flasks/concentration: 2 (blank control, test substance), 1 (inhibition control, reference substance)
- Method used to create aerobic conditions: At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The test assays were stirred using magnetic stirrers.
- Measuring equipment: two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes.
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.

SAMPLING
- Sampling frequency: twice a week
- Sampling method: sodium hydroxide solution for the adsorption of carbon dioxide

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other: reference substance

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

< 10

Sampling time:

28 d

Details on results:

Degree of biodegradation in the inhibition control after 14 days: 28 % CO2/ThCO2

BOD5 / COD results

Results with reference substance:

Degree of biodegradation of the reference substance after 14 days: 77 % CO2/ThCO2

Any other information on results incl. tables

Table 1: Measured data of Total Inorganic Carbon (TIC) [mg/L]

Day		BC NaOH	BC1	BC2	RS	IH	TS1	TS2
0	a	1.2	-	-	-	-	-	-
	b	1.2
3	a	1.3	11.3	17.4	25.7	15.2	15.4	17.9
	b	1.3	11.3	17.4	25.7	15.2	15.4	17.8
6	a	1.4	16.2	22.4	145	143	16.8	20.3
	b	1.4	16.2	22.4	145	144	16.7	20.3
8	a	1.3	16.2	9.8	54.2	37.0	14.1	16.2
	b	1.2	15.6	9.4	53.8	37.0	13.9	16.0
10	a	1.1	15.0	23.3	38.6	23.1	13.2	17.7
	b	1.0	14.9	23.4	38.9	23.0	13.0	17.4
14	a	1.6	25.5	32.9	60.5	46.4	20.0	28.3
	b	1.9	25.2	32.9	60.0	45.4	19.5	27.6
17	a	2.5	20.3	18.1	52.0	26.6	17.1	22.6
	b	2.1	20.2	18.1	51.8	26.0	17.1	22.6
20	a	1.1	19.1	7.5	39.7	20.2	16.4	20.8
	b	1.0	19.1	7.4	39.8	20.3	16.4	21.0
23	a	0.9	17.6	13.0	36.5	20.2	17.9	22.1
	b	0.8	17.7	13.2	36.8	20.1	17.9	22.4
27	a	0.8	19.7	14.8	33.7	23.4	17.7	23.4
	b	0.8	19.6	14.8	33.5	23.2	17.6	23.2
28	a	1.1	8.4	7.8	9.2	11.8	8.7	11.2
	b	1.3	8.4	7.7	9.3	12.0	8.9	11.1
flask 1 29	a		22.9	19.8	6.8	18.1	16.7	16.8
	b		23.0	19.7	6.7	18.1	16.8	17.1
flask 2 29	a		3.5	3.7	2.8	3.8	3.6	3.6
	b		3.7	3.7	3.0	3.6	3.4	3.3

 

 

Table 2: Produced carbon dioxide amount in the test vessels

[mg CO2/test vessel]						mg CO2 added up after subtraction of the mean value of the blank controls
Duration of exposure [days]	BC mv	RS	IH	TS1	TS2	RS	IH	TS1	TS2
3	4.8	9.0	5.1	5.2	6.1	4.2	0.3	0.4	1.3
6	6.6	52.8	52.2	5.7	7.0	50.4	45.9	-0.5	1.7
8	4.2	19.3	13.1	4.7	5.4	65.5	54.8	0.0	2.9
10	6.5	13.7	7.9	4.3	5.9	72.7	56.2	-2.2	2.3
14	10.2	21.7	16.4	6.8	9.8	84.2	62.4	-5.6	1.9
17	6.7	18.7	9.3	5.9	7.9	96.2	65.0	-6.4	3.1
20	4.2	13.9	6.8	5.4	7.0	105.9	67.6	-5.2	5.9
23	4.8	12.6	6.6	5.7	7.3	113.7	69.4	-4.3	8.4
27	5.9	11.9	8.2	6.1	8.2	119.7	71.7	-4.1	10.7
28	2.7	3.1	4.1	2.9	3.8	114.5	72.0	-5.6	10.2
29	8.4	2.8	7.3	6.7	6.8

 

mg CO₂ in the test assay = (TICmv*44*100)/(12*1000)

 

TIC_mv= measured TIC mean value after subtraction of the NaOH blank value

12 = atomic weight of carbon

44 = molecular mass of carbon dioxide

100/1000 = conversion into 100 mL absorption solution

mg CO₂ added up = CO₂mv - CO₂mv BC + CO₂pd

CO₂mv = CO₂ amount in the test assay at sampling day

CO₂mv BC = CO₂ amount in the blank control at sampling day

CO₂pd = CO₂ amount in the test assay at previous sampling day

 

 

Table 3: Theoretical Carbon dioxide [mg/test assay]:

RS : 109.6

IH : 220.7

TS1 : 109.6

TS2 : 110.9

 

Table 4: Degree of Biodegradation [% CO₂/ThCO₂]

Test duration [days]	RS	IH	TS1	TS2	TS mv
0	0	0	0	0	0
3	4	0	0	1	1
6	46	21	0	2	1
8	60	25	0	3	2
10	66	25	-2	2	0
14	77	28	-5	2	-2
17	88	29	-6	3	-2
20	97	31	-5	5	0
23	104	31	-4	8	2
27	109	32	-4	10	3
28	104	33	-5	9	2

 

[% CO2/ThCO2] = (CO2Ta)/(ThCO2Ta* 100)

 

CO₂Ta = CO₂ amount at sampling day

ThCO₂Ta = Theoretical CO₂ amount in the test assay

 

Table 5: Measured values [mg/L], determination of DOC after centrifugation

		[mg/L DOC]
		BC1	BC2	BC mv	RS	IH	TS1	TS2	TS mv
At begin of exposure	a	0.2	0.2	0.2	19.2	-	-	-
	b	0.2	0.2		19.3	-	-	-
At the end of exposure	a	0.9	0.8	0.9	1.1	-	-	-
	b	0.9	0.8		1.1	-	-	-
Degradation of DOC [%]					99	-	-	-	-

 

 

Degradation of DOC [%] = [1 -(CTAt - CBCt)/(CTA0 - CBC0)] * 100

 

CTA_t= average DOC conc. of the reference substance assays; current values

CBC_t= average DOC conc. of the blank control assays; current values

CTA₀= average DOC conc. of the reference substance assays at day 0

CBC₀= average DOC conc. of the blank control assays at day 0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance is not readily biodegradable according to OECD criteria. It is poorly biodegradable.