Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(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:

2016-10-10 to 2016-11-21

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)

Version / remarks:

1992-07-17

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

EC Number:

915-704-1

Molecular formula:

C36H28ClCrN7O10S2.Na / C34H26Cl2CrN8O8S2.Na / C38H30CrN6O12S2.Na

IUPAC Name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

Appearance
- physical state. solid
- color: 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 inoculum/activated sludge: Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 19 October 2016 from the aeration tank of the plant.
- Preparation of inoculum for exposure: A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. 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 test substance concentration 1

Initial conc.:

20 mg/L

Based on:

TOC

Initial test substance concentration 2

Initial conc.:

ca. 44 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: 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; Tte 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: 7.4 ± 0.2
- pH adjusted: yes
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: 2 L incubation bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour
- Measuring equipment: The bottles were connected to 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 closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: s.a.

SAMPLING
- Sampling frequency: twice a week

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no
- Other: Inhibition control, positive control (reference substance, aniline)

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

< 10

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

Degree of biodegradation of the reference substance after 14 days: 85 % CO2/ThCO2

Any other information on results incl. tables

Table 1: Measurement of Total Inorganic Carbon, measured data: TIC [mg/L] 

Day		BC NaOH	BC1	BC2	RS	IH	TS1	TS2
0	a	0.9	-	-	-	-	-	-
	b	0.8
1	a	0.7	6.7	7.0	6.8	6.9	5.1	7.6
	b	0.8	6.9	6.8	6.8	6.8	5.0	7.6
4	a	0.9	24.3	25.1	91.2	109	17.8	23.5
	b	1.0	24.2	25.1	91.2	109	17.5	23.2
7	a	0.8	30.5	30.0	128	115	23.8	21.7
	b	0.7	29.7	29.4	129	116	23.6	21.3
11	a	0.9	37.4	35.6	104	73.3	26.7	27.1
	b	0.8	36.5	35.1	104	73.0	26.2	26.8
14	a	0.8	24.6	25.4	46.4	31.0	20.7	18.8
	b	0.9	23.9	25.1	46.1	31.0	20.5	18.9
18	a	1.2	26.7	25.9	38.2	28.2	23.0	21.3
	b	1.2	26.1	25.4	37.8	27.8	23.1	21.3
22	a	0.8	15.2	17.7	31.2	21.6	24.7	22.0
	b	0.8	15.1	17.7	31.1	21.5	24.8	22.0
25	a	0.9	14.5	15.3	26.0	17.5	19.1	19.4
	b	0.8	14.7	15.1	26.0	17.4	19.0	19.2
27	a	0.7	10.4	12.4	17.3	12.7	14.8	15.3
	b	0.8	10.2	12.3	17.6	12.6	14.6	15.4
28	a	0.8	7.1	7.4	11.0	8.1	8.9	10.3
	b	0.7	7.0	7.3	11.2	8.2	8.7	10.0
flask 1 29	a		15.2	17.6	15.2	15.8	16.3	11.1
	b		15.1	17.3	15.1	16.0	16.4	11.4
flask 2 29	a		4.6	3.7	4.1	4.7	4.6	4.5
	b		4.5	3.7	4.0	4.5	4.6	4.5

 

The NaOH blank value is determined on filling the absorption vessels.

The measured values, for instance applies on day 0 for the first and the second sampling.

The NaOH blank value was subtracted from each test vessel.

 

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
1	2.2	2.2	2.2	1.5	2.5	0.0	0.0	-0.7	0.3
4	8.7	33.2	39.7	6.2	8.3	24.5	31.0	-3.2	-0.1
7	10.7	46.9	42.1	8.4	7.6	60.7	62.4	-5.5	-3.2
11	12.9	37.8	26.5	9.4	9.5	85.6	76.0	-9.0	-6.6
14	8.8	16.7	11.1	7.3	6.6	93.5	78.3	-10.5	-8.8
18	9.2	13.6	10.0	8.1	7.5	97.9	79.1	-11.6	-10.5
22	5.7	11.1	7.6	8.8	7.8	103.3	81.0	-8.5	-8.4
25	5.0	9.1	6.0	6.6	6.6	107.4	82.0	-6.9	-6.8
27	3.9	6.1	4.3	5.1	5.3	109.6	82.4	-5.7	-5.4
28	2.3	3.8	2.7	2.9	3.4	110.7	82.9	-4.9	-6.0
29	6.9	6.5	7.0	7.1	5.2

 

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

  

TICmv = 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: Calculation of degree of biodegradation, Theoretical Carbon dioxide [mg/test assay]:

RS : 109.6

IH : 220.2

TS1 : 111.1

TS2 : 109.9

 

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

Test duration [days]	RS	IH	TS1	TS2	TS mv
0	0	0	1	0	0
1	0	0	-1	0	-1
4	22	14	-3	0	-2
7	55	28	-5	-3	-4
11	78	35	-8	-6	-7
14	85	36	-9	-8	-9
18	89	36	-10	-10	-10
22	94	37	-8	-8	-8
25	98	37	-6	-6	-6
27	100	37	-5	-5	-5
28	101	38	-4	-5	-5

 

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

 CO₂Ta = CO₂ amount at sampling day

ThCO₂Ta = Theoretical CO₂ amount in the test assay

Table 5: Measurement of Dissolved Organic Carbon 

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	0.5	0.4	0.5	18.9	-	-	-
	b	0.5	0.4		19.0	-	-	-
At the end of exposure	a	1.0	0.8	0.9	0.9	-	-	-
	b	1.0	0.9		0.9	-	-	-
Degradation of DOC [%]					100	-	-	-	-

 Degradation of DOC [%] =  {1 - [(CTA_t - CBC_t) / (CTA₀ - CBC₀)]} x 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

List of abrevations:

BC: blank control assay

DIC: Dissolved Inorganic Carbon

DOC: dissolved organic carbon equiv.: equivalent

IH: inhibition control assay

Mv: mean value

RS: reference substance assay

TC: total carbon

ThCO2: Theoretical amount of formed Carbon dioxide

TIC: total inorganic carbon

TOC: total organic carbon

TS: test substance assay

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.