Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(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:

2018-10-02 to 2018-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guideliine

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

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

Constituent 1

Reference substance name:

Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

EC Number:

233-357-8

EC Name:

Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

Cas Number:

10127-27-2

Molecular formula:

C16H11CrN5O8S.Na

IUPAC Name:

sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

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: 2018-09-28
- 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 until test start. Further treatment see section ‘preparation of the test vessels’. 4.55 mL/ L of this mixture were used to initiate inoculation (25.1 mg/L dw).

Colony forming units in the test vessel: approx. 10^7 - 10^8 CFU/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

30 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 – 23.0 °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 adsorption 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 adsorption 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 reference item was 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:

17

Sampling time:

28 d

Details on results:

Based on the carbon content a ThCO2 of 1.26 mg CO2/mg test item was calculated. A test concentration of 30 mg/L, corresponding to a carbon content of 10.3 mg C/L in the test vessels was selected.

Colony Forming Units of the Inoculum
Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 1.62  109 CFU/L, corresponding to approx. 0.74  107 CFU/L in the test vessel.

The adaptation phase of the functional control changed within 2 days into the degradation phase (degradation  10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 8 days and a maximum biodegradation of 84% on day 16. The validity criterion degradation  60% after 14 days is fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 54% was determined within 13 days, with 58% 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 mean of replicates reached the 10% level (beginning of biodegradation) on day 10. The 60% pass level was not reached within the 28 day period of the study. The mean biodegradation on day 28 was 17%.

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

In the inoculum control the total CO2 production was 40.8 mg CO2/L after 28 days.

BOD5 / COD results

Results with reference substance:

In the toxicity control containing both test and reference item a biodegradation of 54% was determined within 13 days, with 58% 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 (batch no.: CHM8004219) 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 2018-10-02 to 2018-10-31, according to OECD 301 B at the test facility. The test item was tested at a concentration of 30 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.3 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 84% on day 16.

 

In the toxicity control containing both test and reference item a biodegradation of 54% was determined within 13 days, with 58% after 28 days. The biodegradation of the reference item was not inhibited by the test itemin the toxicity control.

 

The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. The mean of replicates reached the 10% level (beginning of biodegradation) on day 10. The 60% pass level was not reached within the 28 day period of the study. The mean biodegradation on day 28 was 17%.

 

 

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

 

Biodegradation of the Test Item in Comparison to the Functional Control and Toxicity Control

	Biodegradation [%]
	Study Day [d]
	8	16	22	28
Test Item, 1^stReplicate	7	9	11	14
Test Item, 2^ndReplicate	11	17	17	20
Functional Control	71	84	84	84
Toxicity Control test item + reference item	44	56	57	58