Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(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:

01 November 2017 - 05 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

EC Number:

250-774-0

EC Name:

Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

Cas Number:

31714-55-3

Molecular formula:

C32H19Cl2CrN4O4

IUPAC Name:

[1-{[5-chloro-2-(hydroxy-kappaO)phenyl]diazenyl}-2-naphtholato(2-)-kappaO]{1-[(5-chloro-2-hydroxyphenyl)diazenyl]-2-naphtholato-kappaO}chromium

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Black powder
- Storage conditions: At room temperature

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum: the Secondary effluent of the JRF Sewage Treatment Plant treating predominantly domestic sewage
- Storage conditions and length: not applicable
- Preparation of inoculum for exposure: the secondary effluent was filtered through Whatman No. 1 filter paper
- Pretreatment: the inoculum (as such after filtration) was incubated at test temperature 22 +/- 2 °C for 6 days.
- Initial cell/biomass concentration (after filtration, prior to incubation): 2.74 x 10^4 CFU/mL (Bacteria) and 1.00 x 10^2 CFU/mL (Fungus)

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

5 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: according to OECD 301D
- Preparation of medium: After mixing the required stock solutions into Milli-Q water, the mineral medium was strongly aerated for 20 minutes using aerator and allowed to stand for 20 h at 22 ± 2°C. All operations were performed in a horizontal laminar flow under aseptic condition.
- Test temperature: 22 ± 2°C
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD incubator (Jagkumar & Co. Pvt. Ltd.) with 300 mL BOD bottles
- Number of culture flasks/concentration: 2
- Measuring equipment: Dissolved Oxygen Meter (HQ-40d, HACH-USA)
- Test performed in closed vessels: yes (but not because of significant volatility of the test substance)

SAMPLING
- Sampling frequency: dissolved oxygen concentration on day 0, 7, 14, 21 and 28; nitrate and nitrite concentration on day 0, 7, 14, 21 and 28
- Sampling method:
* dissolved oxygen: DO meter (HQ-40d);
* nitrate and nitrite concentration: 50 and 25 mL sample per time point taken and treated as described in 'details on analytical methods' prior to UV-Vis analysis at 220 nm and 275 nm (nitrate) and 543 nm (nitrite)
- Sample storage before analysis: not indicated

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, containing inoculum only
- Abiotic sterile control: no
- Toxicity control: yes, containing reference substance, test item and inoculum
- Other: procedure control, containing reference substance and inoculum

STATISTICAL METHODS: no statistical methods were used

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.2%

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

4.12

Sampling time:

28 d

Details on results:

- See tables 1, 2 and 3 for more information on dissolved oxygen concentrations, oxygen depletion and dissolved oxygen consumption, respectively.
- The percentage degradation in the toxicity control on day 7 and 14 was 34.97 and 39.51%, respectively. Based on this result, the test item is considered to be not toxic for wastewater microorganisms.
- Nitrate and nitrite content: for both ions the detected absrbance was below the linearity range, thus considered negligible.

BOD5 / COD results

Results with reference substance:

The reference substance degraded for 84.39% and 88.96% after 14 and 28 days, respectively.

Any other information on results incl. tables

The ThOD of the test item was caculated to be 1.943. The ThOD of the reference substance was calculated to be 1.666.

The BOD value for the test item on day 7, 14, 21, and 28 was 0.012, 0.066, 0.076 and 0.080 mg O₂/mg, respectively.

The BOD value for the reference substance in the procedure control on day 7, 14, 21, and 28 was 1.272, 1.406, 1.418 and 1.482 mg O₂/mg, respectively.

Table 1 Mean dissolved oxygen (mg/L)

Treatment	Mean DO (mg/L) on Day
	0	7	14	21	28
Inoculum Blank	8.51	8.14	8.26	8.24	8.23
Procedure Control	8.62	1.89	1.34	1.26	0.93
Test Solution	8.49	8.06	7.91	7.84	7.81
Toxicity Control	8.48	1.80	1.10	-	-

Table 2 Mean oxygen depletion (mg/L)

Treatment	Mean Oxygen Depletion (mg/L) on Day
	7	14	21	28
Inoculum Blank	0.37	0.25	0.27	0.28
Procedure Control	6.73	7.28	7.36	7.69
Test Solution	0.43	0.58	0.65	0.68
Toxicity Control	6.68	7.38	-	-

Table 3 Oxygen consumption (mg/L)

Treatment	Oxygen Consumption (mg/L) on Day
	7	14	21	28
Procedure Control	6.36	7.03	7.09	7.41
Test Solution	0.06	0.33	0.38	0.40
Toxicity Control	6.31	7.13	-	-

Table 4 Percent degradation

Treatment	% Degradation (Based on ThOD)
	7	14	21	28
Procedure Control	76.35	84.39	85.11	88.96
Test Solution	0.62	3.40	3.91	4.12
Toxicity Control	34.97	39.51	-	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'

Interpretation of results:

not readily biodegradable

Conclusions:

The Closed Bottle test showed that T-95/T-99 was not readily biodegradable after 28 days of incubation (4.12% degradation).

Executive summary:

A Closed Bottle test according to OECD guideline 301D and GLP principles was performed to assess the biodegradability of T-95/T-99.

The test item was incubated with inoculum for 28 days at a concentration of 5 mg/L. Secondary effluent from a seawage treatment plant receiving predominantly domestic sewage was used as inoculum after being filtrated. An inoculum blank and a toxicity control were included. Sodium benzoate was used as a reference substance (procedure control). Samples for dissolved oxygen determination were taken on day 0, 7, 14, 21 and 28 and BOD values were calculated. ThOD and BOD values were used to calculate the percentage degradation. The reference item was degraded for 89% after 28 days. The test item was degraded for 4.12% after 28 days. Since the toxicity control showed degradation >25% (39.5%) after 14 days, the test item was considered to be not toxic to wastewater microorganisms.

Based on these results, T-95/T-99 is regarded to be not readily biodegradable. All validity criteria were met and the study was considered to be valid.