Pyridinium, 1-[2-[[4-[[3-[2-[6-amino-1-hydroxy-3-sulfo-5-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-2-naphthalenyl]diazenyl]-4-sulfophenyl]amino]-4-oxobutyl]sulfonyl]ethyl]-3-carboxy-, inner salt, sodium salt (1:4)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 27, 2019 to July 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Dihua sewage treatment plant, Taiwan.

Duration of test (contact time):

29 d

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

1. Before the study starting, prepared inoculum in medium was aerated with CO2-free air for one day.
2. Test grouping:
2.1 Test group: Contain the test article, inoculum and medium. It was conducted with two replicates. In the test, test article (TOC concentration was 21.50%) was added, and total volume was 1.2 L. Final concentration of TOC was 20.00 mg/L.
2.2 Control group: Only contain inoculum and medium. It was conducted with two replicates.
2.3 Reference group: Contain the reference article, inoculum and medium. It was conducted with one replicate. In the test, 4.00g/L reference article (TOC concentration was 29.26%) was added, and total volume was 1.2 L. Final concentration of TOC was 20.00 mg/L.
2.4 Toxicity group: Contain the test article, reference article, inoculum and medium. Total volume was 1.2 L. It was conducted with one replicate. Test article and 4.00 g/L reference article were added, and total volume was 1.2 L. Final concentration of TOC was 40.00 mg/L. (Test article concentration of TOC was 20.00 mg/L and reference article concentration of TOC was 20.00 mg/L).
3. Three absorption bottles, each filled with 0.0125 M barium hydroxide solution of 100mL as CO2 absorbent, were connected in series to each culture flask.
4. Each group was aerated with CO2-free air at rate of 60±10 mL/min. CO2 production was measured on day 2, 5, 7, 9, 14, 19, 23, 28 and 29.
5. During the measurement, an absorption bottle closest to the culture flask was disconnected. The remaining two bottles were moved closer accordingly, followed by a new bottle at the end. The disconnected bottle with phenolphthalein as the indicator was titrated with 0.05 M HCl to determine CO2 production.
6. On day 28, 1.00 mL of concentrated hydrochloric acid (36.5~38%) was added into each culture flask and aerated with CO2-free air for overnight. Level of CO2 production was measured on day 29.

Reference substance:

acetic acid, sodium salt

Remarks:

Sodium acetate

Test performance:

In the test, difference of percentage biodegradation between two replicate of test group were all less than 20.00%. The test was in compliance with validity of the test.

At the end of the test, average cumulative CO2 production of the control group was 66.00 mg/L, which was obviously lower than 70.00 mg/L. This suggested that the test was in compliance with validity of the test

On day 9, percentage biodegradation of the reference article was greater than 60.00%. The test was compliance with validity of the test.

Parameter:

% degradation (CO2 evolution)

Value:

< 60

Sampling time:

29 d

Details on results:

In the study, average percentage biodegradation (% ThCO2) of test groups were 1.06%, 0.87%, -0.19%, -1.00%, -2.81%, -5.37%, -7.30%, -9.98% and -11.79% on day 2, 5, 7, 9, 14, 19, 23, 28 and 29, respectively.

Percentage biodegradation (% ThCO2) of the reference group were 21.73%, 46.95%, 59.43%, 66.30%, 72.79%, 76.29%, 77.73%, 77.48% and 78.41% on day 2, 5, 7, 9, 14, 19, 23, 28 and 29, respectively.

Percentage biodegradation (% ThCO2) of the toxicity group were 11.17%, 23.27%, 29.90%, 32.83%, 52.02%, 35.20%, 34.49%, 33.05% and 32.21% on day 2, 5, 7, 9, 14, 19, 23, 28 and 29, respectively.

The pH of all groups on day 0, 28 and 29 were range between 7.62~7.67, 7.58~8.57 and 2.05~2.21.

Results with reference substance:

At Day 14 during the study, the biodegradation was 72.79 %.

Table 1. The CO₂ production of control group, test group, reference group and toxicity group

Time (Day)	Control group (mg) (1) (2) Mean			Test group (mg) (1) (2)		Reference group (mg)	Toxicity group (mg)
2	5.39	4.51	4.95	6.49	5.28	24.09	24.64
5	7.70	7.92	7.81	8.69	6.60	30.03	29.15
7	5.61	7.37	6.49	6.27	4.84	17.49	18.15
9	4.29	5.17	4.73	4.29	3.74	10.78	9.90
14	7.70	10.34	9.02	7.81	7.04	14.74	12.87
19	9.90	11.88	10.89	9.13	8.14	13.97	11.22
23	10.78	12.65	11.72	10.45	9.57	12.98	10.45
28	11.00	12.98	11.99	10.12	9.13	11.77	9.46
29	10.67	12.54	11.61	10.67	9.35	12.43	10.12

Table 2. The cumulative CO₂production and percentage biodegradation of control group, test group, reference control and toxicity group

Time (Day)	Cumulative CO2production (mg)  Control group (mg)  Test group (mg) Reference Toxicity Mean (1) (2)  group  group					Percentage b odegradation (% ThCO2)  Test group (mg) Reference Toxicity Mean (1) (2)  group  group
2	4.95	1.54	0.33	19.14	19.69	1.75	0.37	1.06	21.73	11.17
5	12.76	2.42	-0.88	41.36	41.03	2.75	-1.00	0.87	46.95	23.28
7	19.25	2.20	-2.53	52.36	52.69	2.50	-2.87	-0.19	59.43	29.90
9	23.98	1.76	-3.52	58.41	57.86	2.00	-3.99	-1.00	66.30	32.83
14	33.00	0.55	-5.50	64.13	61.71	0.62	-6.24	-2.81	72.79	35.02
19	43.89	-1.21	-8.25	67.21	62.04	-1.37	-9.36	-5.37	76.29	35.20
23	55.61	-2.48	-10.40	68.48	60.78	-2.81	-11.79	-7.30	77.73	34.49
28	67.60	-4.35	-13.26	68.26	58.25	-4.93	-15.04	-9.98	77.48	33.05
29	79.20	-5.28	-15.51	69.08	56.76	-5.99	-17.60	-11.79	78.41	32.21

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

According to OECD 301B test method, CR SB33 was not be considered readily biodegradable.

Executive summary:

This test using the procedures outlined in the SuperLab Study Plan for MZ6-181200029 which is based on the SOP for the OECD 301B (SOPE-503) and OECD 301B (OECD, 1992). The results of this OECD 301B test for CR SB33 show that all validity criteria were met. CR SB33 can not be considered readily biodegradable under the test conditions and the percentage biodegradation of CR SB33 in sludge was less than 60 %.

Description of key information

Not readily biodegradable (OECD TG 301B).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

According to analysis of total organic carbon, TOC result of CR SB33 was 214,950 mg/kg. Therefore, the TOC concentration of CR SB33 was 21.50%. Difference of percentage biodegradation between two replicate of test group were all less than 20.00%. The test was in compliance with validity of the test. Average cumulative CO₂ production of the control group was 66.00 mg/L, which was obviously lower than 70.00 mg/L. This suggested that the test was in compliance with validity of the test. On Day 9, percentage biodegradation of the reference article was greater than 60.00%. The test was compliance with validity of the test. Percentage biodegradation (% ThCO₂) of CR SB33 were all <60.00%. Therefore, CR SB33 was not the readily biodegradable article.